Abstract. Kumar R, Tapwal A, Pandey S, Borah RK, Borah DP, Borgohain J. 2013. Macro-fungal diversity and nutrient content of
some edible mushrooms of Nagaland, India. Nusantara Bioscience 5: 1-7. The northeast region of India abounds in forest wealth,
including variety of flora and fauna. The high humidity during monsoon period provides ideal atmospheric conditions for the growth of
diverse group of macrofungal fruit bodies. Nagaland, the northeastern state of India is rich in biodiversity and encompasses large
numbers edible and non-edible mushroom species. Young and matured carpophores of 15 wild edible mushroom species were collected
from 12 locations in different districts of Nagaland. Out of these four species belongs to family Agaricaceae, two belongs to
Tricholomataceae and rest belongs to Boletaceae, Cantherallaceae, Russulaceae, Sarcoscyphaceae, Auriculariaceae, Polyporaceae,
Schizophyllaceae, Pleurotaceae and Lyophyllaceae. The selected species were analyzed for proximate analysis of nutritional values. The
protein content varies from 22.50-44.93% and carbohydrates were recorded 32.43-52.07% in selected species. The documentation of
wild edible mushrooms is very scanty in Northeast India. The key objective of the present study was to generate a database on
macrofungal diversity, ecology, ethnomycology, utilization and nutrient status of important wild edible mushroom species of Nagaland,
which forms a part of the food culture of the native peoples.
Key words: Proximate analysis, carpophores, ethnomycology

INTRODUCTION
Mushrooms have been the objects of much curiosity
and speculation since time immemorial. They are one of
the most important components of the forest ecosystem.
Their edibility, poisonous nature, psychotropic properties,
mycorrhizal and parasitic associations with the forest trees
make them economically important and interesting to
study. The northeast region of India abounds in forest
wealth, including many species of trees and other woody
plants. The biodiversity of woody flora is correlated with
an equally diverse mycoflora. The high humidity during
monsoon period provides ideal atmospheric conditions for
the growth of many saprophytes, including the mushrooms.

There are many mushrooms growing in the forests of
Nagaland and local relish on them. They have diverse
shapes, sizes and colors and also have varied appearance,
ranging from patches on wood to brackets, coral-like tufts
simple clubs rosettes cauliflower like structure or centrally
or laterally stalked fruit bodies. Mushrooms can be
categorized as edible or non-edible. The poisonous effects
of mushrooms were dealt with in an epigram written by
Euripides in about 450 B.C (Giovanni 1989). Right from
the beginning man has learnt to differentiate the edible and
non edible mushroom through numerous observation, trials
and errors. Through these experiences man has learned to
use mushrooms as a part of their diet. Seasonal mushroom
hunting and collection are the part of seasonal activity of

2

5 (1): 1-7, May 2013

the people. Barros et al. (2008) reported the wild
mushrooms are richer sources of protein and have a lower
amount of fat than commercial mushrooms. The proteins of
wild edible mushroom contains considerable amounts of
non-essential amino acids like alanine, arginine, glycine,
glutamic acid, aspartic acid, proline and serine (Manzi and
Pizzoferrato 2000).The add-value arising from mushrooms
are bioactive materials which lead to an increase in its
consumption
and
therefore,
stimulating
the
commercialization of edible species. Mushrooms also have
been used extensively in traditional medicine for curing
variety of diseases including viral infection, bacterial
infection, cancer, tumor, inflammation, cardiovascular
diseases (Benedict and Brady 1972; Iwalokum et al. 2007)
Many researchers have been working on wild
mushroom and reported more than 2000 species of edible
mushroom all over the world (Adhikari 2000; Purakasthya
& Chandra 1985) have reported 283 edible species from
India, out of which some are cultivated. Production of
mushroom all over the world exceeds three million tones.
Most of the exporting countries are Netherlands, Poland,
Ireland, Belgium, India and China. Among these countries
China is the largest exporter of preserved mushrooms. In
India most commonly cultivated mushroom species are
Button (Agaricus bisporus), Oyster (Pleurotus spp.) and
Paddy straw mushroom (Volvariella volvacea) as
documented by (Harsh and Joshi 2008).In India, mushroom
is a unique non-traditional cash crop and as popular as food
among the tribal people of north east India. Many rural
communities of Nagaland are using mushrooms in their
traditional dishes because of their delicious flavor. The
favorable climatic condition of north-eastern states of India
leads to rich mushroom diversity and form a valuable nontimber forest resource for local folk. Mushrooms are sold in
traditional markets or commercially exploited as food or
medicines (Tanti et al. 2011). Some of the edible species
like Termitomyces
eurrhizus, Lentinus conatus,
Schizophyllum commune, Tricholoma giganteum and
Pleurotus are sold in the markets of Kohima district of
Nagaland by the local people (Tanti et al. 2011). In-spite of
rich diversity of mushrooms in Nagaland state very few
studies have been reported on diversity and market survey
from North-Eastern Hills of India ( Verma et al. 1993;
Singh et al. 2007 and Sarma et al. 2010).
The main objective of the present study was to generate
a database on ecology, ethnomycology, utilization and
nutrient status of important wild edible mushroom species
of Nagaland, which forms a part of the food culture of the
Nagaland people.
MATERIALS AND METHODS
Study area
Nagaland is situated northeastern part of India having
longitude of 93°20´ E to 95°15´ E and Latitude 25°6´ N to
26°4´ N and having eleven districts with 16,579 Km2 area.
The forest cover is about 86% including reserve forests.
The prominent tribes of Nagaland are Chakhesang, Angami,
Zeliang, Ao, Sangtam, Yimchunger, Chang, Sema, Lotha,

Khemungan, Rengma, Konyak, Pachury and Phom. The
average annual rain fall ranges from 2000-2500 mm and
average temperature during the summer ranges between 15
to 30ºC and in the winter it can fall below to 4ºC.
Sample collection and diversity analysis
The periodic surveys were made to Lahorijan, Puliebzie,
Zakhama, Pherma, Mankoi, Chungtia, Nongkham, Namcha,
and Tigit forest for the collection of macrofungi during
rainy season (June to September) and winter (October to
December) in 2010-2011. The collected samples were
wrapped in wax paper and brought to the laboratory for
identification and proximate analysis. The taxonomy has
been worked on the basis of macro and microscopic
characteristic following available literatures (Zoberi 1973;
Alexopolous et al. 1996; Purakasthya 1985 and Singer
1986). The soft textured specimens were preserved in 2%
formaldehyde and leathery textured were preserved in 4%
formaldehyde and kept in museum of Forest Protection
Division, Rain Forest Research Institute, Jorhat, Assam by
assigning identification number. The traditional knowledge
on the wild mushrooms were gathered from the local tribes
and used to know the edibility and medicinal value. The
frequency and density of different species has been
determined by the following formulas:
No. of site in which the sp. is present
Freq. of fungal sp. (%) = ----------------------------------------- x 100
Total no. of sites
Total no. of individual of a particular species
Density = ------------------------------------------------------- x 100
Total no. of species
For proximate analysis, the fruit bodies were oven dried
and powdered in a Moulinex blender. The fine powdered
samples were stored in the desiccators and utilized for
proximate and mineral nutrients analysis following
Anthrone method (Fasidi and Kadiri 1993).
Moisture content: The fresh and oven dried weight
(80°C for 48h) of each mushroom species was recorded
moisture content was determined (Raghuramulu et al.
2003) by formula:
Fresh weigh – dry weight
Moisture content (%) = -------------------------------- x 100
Fresh weigh
Dry matter content: Weight obtained after oven drying
at 80°C for 48 h.
Crude fiber: The Crude fibers content was calculated as
following equation:
Crude fiber (g/100 g sample) = [100 − (moisture + fat)]
× (We-Wa)/Wt of sample (Raghuramulu et al. 2003).
Protein content: 0.5 g of the powdered mushroom
sample was extracted with 50.0 cm of 2% NaCl in a waterbath at 60°C for 1 h. The extract was filtered out and 50.0
cm of 3% copper acetate monohydrate were added to the
filtrate to precipitate protein. The precipitated protein was
then centrifuged out and dissolves in 50 cm of 0.1 m
NaOH. The quantity of protein in the alkaline solution was

Cookeina sulcipe (Berk.) Kuntze. It grows as
saprotrophs on dead wood, fruiting bodies cup-shaped to
funnel-shaped, brightly-colored, and yellow to red. The
outer surface is less brightly colored the walls of the
apothecium, is thin and flexible and has tiny hairs on the
upper rim of the cup, asci are constricted abruptly below
and form a blunt, rounded base with a slim, tail-like
connection, ascospores ellipsoidal and smooth 20-40.5µm
long (Figure 1K, 2K ).
Schizophyllum commune (Fr. Gemeiner Spaltblättling).
It grows in dead wood of deciduous trees, fruiting body 1-5
cm wide, fan-shaped small hairs on the upper surface,
white to grayish, stem rudimentary or absent, gills Under
surface of the fruiting body composed of gill-like folds in
the undersurface that are distinctively split, spore
Cylindric, 5x3um, cystidia absent spore print White (Figure
1B, 2B).
Lepista irina (Fr.) H.E. It’s found in open woodland,
cap light brown, 5-11cm across, flattened-convex, wavy at
the margin, stem 55-97x8-20mm, dirty white, covered in
long fibres, ochraceous near the base, gills emarginate,
crowded, spores oval,7-9x3.5-4µm, spore print dirty pink
(Figure 1G, 2G).
Melanoleuca grammopodia (Bull.) Murrill. It found in
woods on leaf mulch and on composted soil, cap convex,
then flattened, with a broad central bump, often depressed,
smooth, gills broad, emarginate, whitish, or cream, stem
equal with a broad base, whitish, with brown fibres along
the length, spores ellipsoidal, smooth 8.5-9.5x 5-6 µm,
basidia four spored, spore print white (Figure 1N, 2N).
Species diversity of macrofungi is related to the
particular habitats. The factors like geographic location,
elevation, temperature, humidity, light and surrounding
flora greatly influence the growth and development of
macrofungi. In present study, the fungal fruitbodies were
collected from 12 different locations of nine districts of
Nagaland. 15 species of edible mushrooms were found, out
of which 4 belongs to family Agaricaceae, 2 belongs to
Tricholomataceae, and one each in Boletaceae,
Cantherallaceae, Russulaceae, Sarcoscyphaceae, Auricula-

riaceae, Polyporaceae, Schizophyllaceae, Pleurotaceae and
Lyophyllaceae. The diversity analysis revealed that
maximum frequency occurrence was exhibited by
Auricularia auricula-judae (66.6%), followed by Agaricus
langei and Lactarius hygrophoroides (58.3% each),
Pleurotus pulmonarius (50%) and minimum (16.6%) by
Melanoleuca grammopodia. The rest of species exhibited
the frequency of distribution between 16.6-50%. All of the
selected species are edible and among which four have
medicinal importance also (Table 1). Recently, Tanti et al.
(2011) has recorded 13 number of macrofungi under 9
genera and six families available in the market of Kohima
town of the Nagaland.
Mushrooms are delicious food due their high quality
protein, vitamins and minerals. The proximate composition
of the selected edible mushroom species has been presented
in Table 2. Fresh mushrooms contained about 90%
moisture and 10% dry matter and dry mushrooms
contained about 90% dry matter and 10% moisture (Chang
and Buswell 1996). In the present study it was observed
that the moisture content of the collected mushroom
samples ranges from 52.11-95.13%. The Pleurotus,
Agaricus and Lepiota have higher moisture content in
comparison to other species. The dry matter content ranged
from 2.1-4.2% with exception to S. commune, having
12.9% dry content. Crude fibres were recorded minimum
for A. arvensis (0.14%) and maximum 12.9% for H.
tessulatus, rest were in between. Edible mushrooms are
highly valued as a good source of protein and their protein
contents usually ranges from 28.93% to 39.1% of dry
weight (Ragunathan et al. 2003; Sanmee et al. 2003).
Following similar trend, the highest protein content was
recorded for L. hygrophoroides (44.93%) and lowest for S.
commune (22.50%). The carbohydrates content of edible
mushrooms usually range from 40.6% to 53.3% of dry
weight (Khanna et al. 1992; Ragunathan et al. 1996). In the
present study, species have carbohydrates between 32.4352.07%. The ash content has exhibited quite variation from
0.18-14.97% in different species.

CONCLUSION
The identification and use of wild edible mushrooms
play a vital role in enrichment of the socio-economic life of
the tribal people. The current environmental issues of
global warming and climate change would adversely affect
the regeneration and growth pattern of the delicate fungi
which requires a specific micro-climate. Consequently, the
high nutritional quality and unique flavor of these
mushrooms are likely to be lost if these wild edibles are not
properly documented. However, a through screening is
needed to delimit their different medicinal properties which
will not only help in solving the food crisis which is
prevalent in the rural poor population but will also add
medicinal touch to their food.
ACKNOWLEDGEMENTS
The authors are gratefully acknowledged to Indian
Council of Forestry Research and Education (ICFRE) for
funding the research project: No-RFRI-39/2010-11/FP.
REFERENCES
Adhikari MK. 2000. Mushrooms of Nepal. P. U. Printers, Kathmandu.
Alexopoulos CJ, Mims CW, Blackwell M. 1996. Introductory Mycology.
4th ed. John Wilay and Sons, New York.
AOAC. 1990. Official methods of analysis of Association of Official
Analytical Chemist. 15th ed. AOAC.Washington DC.
Benedict RG, LR Bradly. 1972. Antimicrobial activity of mushrooms
metabolites. J Pharm Sci 61: 1820-1822.
Chang ST, Buswell JA. 1996. Mushroom nutraceuticals. World J
Microbiol Biotechnol 12: 473-476.
Fasidi IO, Kadiri M. 1993. Effect of sporophores maturity on chemical
compoisiton of Volvariella esculenta (Mass) Singer, a Nigerian edible
mushroom. Die Nahrung 37: 269-273.

Abstract. Singh RK, Malik N, Singh S. 2013. Impact of rhizobial inoculation and nitrogen utilization in plant growth promotion of
maize (Zea mays L.). Nusantara Bioscience 5: 8-14. During the course of growing population demands there has been an increasing
interest in exploring the possibility of extending the beneficial interaction between cereals and plant growth promoting rhizobacteria
(PGPR). Endophytes are a group of microorganism that resides mostly in the intercellular space of various parts of plants including
cereals. Assessment of plant growth promoting properties of the five-rhizobial strains belonging to α subclass i.e. Rhizobium
leguminosarum bv. phaseoli RRE6 and R. undicola RRE36 and those belonging to β subclass i.e. Burkholderia cepacia (RRE3, RRE5,
RRE25) was done by growing maize plants inoculated with these strains. Inoculated maize plants showed a significant increase in plant
height, root length, shoot and root dry weight over uninoculated control. R. leguminosarum bv. phaseoli RRE6 and B. cepacia RRE5
among the α and β-subclass representatives respectively, gave the best inoculation response. Effect of nitrate supplementation upon
maize-RRE6 and RRE5 association was also studied and a significant increase in all the growth parameters and colonization ability was
recorded when nitrate was present as a supplement over uninoculated control and maize-RRE6 and RRE5 in absence of external nitrate.
Key words: Rhizobium leguminosarum bv. phaseoli, R. undicola, B. cepacia, Zea mays, nitrate utilization

INTRODUCTION
PGPR are plant growth promoting rhizobacteria that
directly or indirectly induce beneficial effects on plant
growth and development. They can occur naturally as
rhizospheric, endophytic or symbiotic component of
bacteria plant association. PGPR can affect plant growth
and development either indirectly or directly. Direct
mechanisms include production of phytohormones,
synthesis of 1 aminocyclopropane-1-carboxylate (ACC)
deaminase, phosphorous solubilization, nitrogen fixation
and siderophore production. Some indirect mechanisms are
that they act as biocontrol agents and induce systemic
resistance in plants (Ashraf et al. 2013). Occurrence of
endophytic association has been reported in many non

leguminous crops such as maize, wheat, millets, kallar
grass, sugarcane, etc. (Webster et al. 1998, Chaintreuil et
al. 2000; Gutierrez-Zamora and Martinez-Romero 2001;
Matiru and Dakora 2004). Plant-microbe interactions may
occur at phyllosphere, endosphere and rhizosphere
(Bhattacharyya and Jha 2012).
Maize (Zea mays) is widely cultivated throughout the
world and it is one of the most important staple grain crops
in the world. Nitrogen and phosphorus are two of the
essential nutrients for maize plant growth and development.
Large quantities of chemical fertilizers are used as to
replenish soil N and P. Use of high levels of nitrogenous
fertilizers in crop production has its drawbacks. Only onethird of the nitrogen applied as a chemical fertilizer is used
up by the crop. The non-assimilated nitrogen results in

SINGH et al. – Impact of rhizobium and nitrogen in growth of maize

nitrate (NO3-) contamination of ground water supplies
(Mytton 1993; Shrestha and Ladha 1998), a potential health
hazard; soil acidification (Kennedy and Tchan 1992) and
increased
denitrification.
Soil
acidification
and
denitrification results in high emission of nitrous oxide
(N2O), a potent greenhouse gas, which enhances global
warming (Bronson et al. 1997). Thus we are in dire need of
exploring alternate or supplementary non-polluting sources
of N for agriculture (Ladha et al. 1997). This problem
could be solved if maize and other cereals were able to
establish more intimate associations with plant growth
promoting microorganisms. It, therefore, would be a
noteworthy achievement if maize could profit from
biological nitrogen fixation thereby decreasing its
requirement and dependence on chemical nitrogenous
fertilizers (Chelius and Triplett 2000; 2001). We have
therefore endeavoured to see if inoculation of some
endophytic bacteria can improve growth performance of
maize.
MATERIALS AND METHODS
Details of bacterial strains used in the study are listed in
Table 1. Spontaneos mutants of Burkholderia strains
RRE3, RRE5, RRE25, and Rhizobium strains RRE6 and
RRE36 were isolated which were resistant to various
antibiotics to be used as genetic marker during the study.
Table 1. Bacterial strains used in the present study
Strains
Burkholderia cepacia RRE3
B. cepacia RRE25
B. cepacia RRE5
Rhizobium leguminosarum bv. phaseoli RRE6
R. undicola RRE36

Accession
number
AY 946010
EU 246850
AY 946011
AY 946012
EU 512923

Characterization of the strains
For growth studies and utilization of different nitrogen
sources all the bacterial strains were inoculated in Yeast
Extract Mannitol (YEM, Vincent 1970) and grown upto
109 cells ml-1 and Rhizobial Minimal Medium (RMM) for
specific experiments (Diebold and Noel 1989). Sodium
glutamate of minimal medium was replaced by nitrogen
sources like sodium nitrite, sodium nitrate and ammonium
sulphate (10mM each) to study nitrogen utilization. Indole
acetic acid (IAA) quantification and siderophore
production were assayed as described by Patten and Glick
2002; Penrose and Glick 2003. Phosphatase enzyme
activity was studied using Pikovskaya’s broth medium.
Pectinase assay was done according to the method of
Mandels (1985). To estimate nitrogenase activity the
acetylene reduction assay of Stewart et al. (1967) was used.
Plant growth experiment (green house conditions)
To study the effect of inoculation with different
antibiotic marked endophytic strains on growth of maize

9

plant, seeds of the maize cultivar Malaviya 1 were selected
and surface sterilized following standard protocol
according to Singh et al. 2006. Three days old maize
seedling, with root length ranging from 2.0 cm to 3.0 cm,
were inoculated separately with one ml each of
exponentially grown bacterial culture having cell
population of 109 CFU (colony forming units) ml-1.
Uninoculated seedlings served as control. The germinated
maize seedlings were transferred aseptically to plastic pots
containing sterile sand. Sand was washed three times with
tap water and dried in hot air oven. Washed and dried sand
was then autoclaved twice for 20 minutes at 120 °C with an
interval of 24 hours. All treatments were arranged in 25
pots i.e. 5 replicates with 5 pots per replication. The plants
were incubated in plant growth chamber under a
combination of fluorescent and incandescent light with a
light intensity of 16000 lux, with a cycle of 16 hrs light and
8 hrs dark cycle temperatures of 28°/23°C and relative
humidity of 55/75%. Plants were regularly watered with
Nitrogen free Fahraeus medium (NFM, Fahraeus 1987) and
were harvested at 35 days after inoculation.
Re-isolation and characterization of putative
endophytes
Root portion of uprooted maize plants were cut into
small pieces of 5 mm length. Surface sterilization was done
twice once with 95% ethanol and then with 0.2% acidified
HgCl2. Sterilized root pieces were macerated in 10ml
sterilized distilled and were decimally diluted (10-5) and
spread on YEM agar plates supplemented with appropriate
antibiotics.
Growth of maize seedling as influenced by nitrate
availability and PGPR inoculation
Two antibiotic resistant derivatives of endophytic
rhizobia, i.e., Rhizobium leguminosarum bv. phaseoli
RRE6strR (resistant to streptomycin 100μg/ml) and
Burkholderia cepacia RRE5strR (resistant to streptomycin
500μg/ml) were used for this study. Surface sterilized seeds
of maize (Malaviya 1) were germinated in Petri plates
containing moistened filter paper under aseptic conditions.
Three-day old seedlings, with root length ranging from 2 to
4 cm, were selected and soaked in 25 ml of bacterial
suspension of RRE-5strR and RRE-6strR for 90 min.
Uninoculated seedlings served as control. Soaked seedlings
were then removed and transferred to agar-water plates
supplemented without and with 10mM NO3-. Four
seedlings were planted on each Petri plate and five
replicates were considered for each treatment. These
seedlings were allowed to grow for 48 h and then removed
from agar water plates and washed in distilled water in
order to remove the agar adhering to the roots. Shoot
length, root length and fresh weight were recorded. Two
grams of root were taken from each treatment separately
and macerated. Macerate was decimally diluted, 100 μl of
each of these solutions was spread on streptomycincontaining YEM plates. Subsequently, the plates were
incubated at 28°C and CFU were counted. For analysis of
data, the completely randomized design was used.

10

5 (1): 8-14, May 2013

to be more effective. A
significant increase in shoot dry
weight was observed in maize
plants inoculated with all
endophytic rhizobial isolates.
Maximum shoot dry weight was
observed in plants inoculated
with R. leguminosarum RRE6
followed by R. undicola RRE36
(Table 2). Shoot dry weight was
found to be more in the case of
Rhizobium
strains
when
compared with Burkholderia
strains.
A significant increase in root
dry weight was exhibited by all
the bacterial endophytes. The best
Figure 1. Maize plants inoculated with endophytic bacterial isolates. Note: 1. Control
performance for dry weight was
(uninoculated), 2. R. undicola RRE36, 3. B. cepacia RRE3, 4. R. leguminosarum RRE6
shown by R. leguminosarum
RRE6 followed by R. undicola
RRE36. When Burkholderia
Statistical analysis
Data related to plant growth parameters were subjected strains were considered, significant increase over the
to analysis of variance using SPSS software. Treatment control was apparent (Table 2). The cell densities from the
means were compared at 95% and 99% probability level (P sterilized root macerates of uprooted maize plants were
= 0.05 and 0.01), and the same set of data was further calculated and found to be similar in each case (Table 3).
analyzed to calculate the least significant difference at P =
Table 2. Effect of inoculation of endophytic rhizobia on
0.05 and 0.01, respectively.
promotion of growth of maize plant (green house conditions)

RESULTS AND DISCUSSION
Response of bacterial inoculation on the growth of
maize plant under green house conditions
The maize cultivar Malaviya 1 was used in plant growth
experiment test to study the effect of inoculation of various
endophytic rhizobial isolates on its growth. The statistical
analysis showed that there was significant effect of
inoculation over the control (uninoculated). Statistically
significant differences were also observed among the
isolates. Significant increase in plant height was observed
in all rhizobial treatments (Figure 1). R. leguminosarum
RRE6 gave the best inoculation response with maize and
resulted in maximum increase in plant height, whereas B.
cepacia RRE3 gave the minimum plant height increase
over the control (Table 2). R. undicola RRE36 also showed
a very good response to inoculation in terms of plant
height. When strains were compared among themselves, B.
cepacia RRE5, R. undiocola RRE36 and B. cepacia RRE3
differed significantly from each other.
Insofar as root length is concerned, it was found that
there was significant increase over the control in the cases
of R. leguminosarum RRE6, B. cepacia RRE5 and R.
undicola RRE36. But in cases of B. cepacia strains RRE3
and RRE25, differences were statistically non significant
(Table 2). In the case of RRE6, there was a significant
increase compared to the control (60% increase) due to
inoculation. RRE36 also showed a significant increase
when compared with the control plant. Statistically
significant difference was observed between both
Burkholderia strains (RRE5 and RRE25); RRE5 was found

Biological activity of the rhizobial endophytes
All the 5 endophytes i.e. R. leguminosarum bv. phaseoli
RRE6, R. undicola RRE36, B. cepacia (RRE3, RRE5,
RRE25) grew well in the presence of different nitrogen
sources (Table 4). It was found that all the strains were
similar in their ability to produce auxin, at an average of
3.00 μg ml-1 (Table 5). The strains were also able to
solubilize phosphate and pectin (Figure 2a, b). Nitrogenase
activity was found to similar in all the strains. The
production of siderophores by bacterial endophytes was
positive as detected on chrome azurol S (CAS) plates.
Growth of all the strain was found to be identical in both
rich medium and minimal medium.
From the present observations it was clear that among
the α and β subclass representatives, R. leguminosarum
RRE6 and B. cepacia RRE5 were the most effective in
increasing different growth parameters of maize. The
present results clearly indicates that the α-subclass
representatives were better growth enhancer as compared
to β-subclass ones, though representatives of both the
classes showed significant increase in the growth when
compared to the control or uninoculated plants. For further
study on these aspects, B. cepacia RRE5 and R.
leguminosarum RRE6 were used to compare between
Burkholderia and Rhizobium.
Table 4. Utilization of nitrogen sources by rhizobial strains in
minimal medium (MM)
Strain
RRE3
RRE36
RRE25
RRE5
RRE6

Effect of nitrate availability and PGPR inoculation on
the growth of maize seedlings
In this experiment, observations were recorded 48 h
after inoculation. In the presence of nitrate, the growth of
maize seedling was enhanced significantly upon
inoculation with both the strains (Figure 3). The combined
effect of nitrate enrichment and strains was much more
significant than the individual effects of these two
variables. Root length, shoot length and fresh weight
significantly increased in all the cases when compared to
the control (uninoculated) plants. Effect was more
pronounced in R. leguminosarum RRE6 than in B. cepacia
RRE5 when nitrate was added as supplement (Table 6).
Number of bacterial colonies recovered from root
macerates of inoculated plants of maize
The number of endophytes recovered from the surfacesterilized roots in case of R. leguminosarum RRE6 was
found to be 3.39 x 106, which was higher than B. cepacia
RRE5 (2.35 x 106). In both the cases, nitrate addition
increased the number of colonies and in case of
RRE6+NO3- it was more than that in RRE5+NO3- (Table
7).

Discussion
Main objective of this study was to demonstrate the
effect of bacterial strain on maize genotype that provide
increased plant productivity compared with the
uninoculated control under fully sterilized conditions.
Representatives of α subclass of Proteobacteria (R.
undicola RRE36 and R. leguminosarum RRE6) and β
subclass of Proteobacteria B. cepacia (RRE5, RRE3 and
RRE25) were used for the plant growth promotion
experiment. Results clearly indicated a significant increase
in various plant growth parameters in presence of the above
strains when compared to the control or uninoculated plant.

The most important parameter studied in this experiment
was dry weight. When dry weight was considered both
shoot and root dry weight were increased significantly
when inoculated with the different endophytic strains. A
similar association was described between maize and
Rhizobium etli (Gutierrez-Zamora and Martinez-Romero
2001). The later authors reported an increase in maize plant
dry matter upon R. etli inoculation. PGPR strains
Pseudomonas and Bacillus significantly affected the height
and dry weight of maize plants as was found by Jarak et al.
(2012).
PGPR can enhance the growth and development of
associated crops by improving nutrient uptake (Biswas et
al. 2000). Shoot growth increased several folds in the
present experiment..The exact mechanism of plant growth
promotion by these isolates is not well understood in the
case of maize. Phytohormone production, phosphate
solubilization, nitrogen fixation and certain phenotypic
changes like root proliferation could be the possible ways
through which the host plants were benefited. PGPR uses
one or more mechanisms to improve the growth and health
of plants and can act simultaneously or independently at
different stages of plant growth. Among these,
phosphosolubilization, nitrogen uptake, and phytohormone
production (indole-3-acetic acid), pectin solubilization was
found to be present in all the strains. Large proportion of
phosphorus in soil is insoluble and therefore unavailable to
plants and hence phosphate solubilization is a desired
property to be present in the bacteria. All tested rhizobial
endophytes were able to solubilize phosphates and act as
PGPR. They also act as biological agents through the
production of siderophores.
Rhizobium-cereal associations are quite dynamic,
enhancing the plant’s root architecture as well as the
overall growth physiology. This finding suggests that
endophytes get intimately associated with roots of maize
seedling in a very early stage of development of plant.
Nitrate uptake and root architecture are affected by PGPR
and nitrate availability. Nitrate transporter genes are
induced by the presence of external nitrate which elicits
root elongation and biomass. The effects of PGPR on

SINGH et al. – Impact of rhizobium and nitrogen in growth of maize

nitrate uptake are similar to those of low nitrate availability
(Mantellin et al. 2003). Changes in root architecture similar
to those induced by PGPR are due to the changes in nitrate
availability in the medium (Wiersum 1958). In the present
study, it was found that in the presence of nitrate (10mM)
along with the strains, the growth of maize seedling was
enhanced. It was found to be the highest in the case of R.
leguminosarum RRE6, supplemented with nitrate. Alami et
al. (1999) found that Rhizobium could be used in
association with non-legume crops to better utilize the
nutrients. The ability to utilize various nitrogenous
compounds by rhizobial endophytes for their growth may
be correlated with their evolutionary history. Due to
extensive use of nitrogenous fertilizer in field, only those
microorganisms could survive who had an inherent
capacity to utilize the diverse nitrogenous compound. In
the present experiment, it has been found that rhizobial
inoculation to maize cultivar Malaviya 1 resulted in
proliferated root architecture.
When nitrate was added as the supplement, growth was
found to be more significant in all the cases (Table 6).
Endophytic rhizobia may alter the morphological and
physiological development of maize plant in ways that
make them better miners of the existing resources of plant
nutrients in soil. This has been reported in previous studies
showing significantly increased production of root biomass
in plants inoculated with certain rhizobial endophytes
(Yanni et al. 1997; Praytino et al. 1999; Biswas et al. 2000;
Yanni et al. 2001). It was found that maize seedling roots
become intimately associated with bacterial cells in 48 h.
During this initial period it was found that there was
significant increase in all the growth parameters studied
and endophytes recovered from the surface-sterilized roots
in case of R. leguminosarum RRE6+NO3- were found to be
6.79 x 106, which is higher than B. cepacia RRE5+NO3(5.72 x 106). Earlier, it was always thought that rhizobia
did not enter non leguminous root tissues in any substantial
manner (Sprent 1989). In the present study it was found
that bacterial population obtained from nitrate enriched
root macerates of maize was very high in both the strains.
So it might be possible that these endophytes are entering
inside the roots of maize seedling.
CONCLUSION
Both the bacterial groups, i.e., Burkholderia cepacia
RRE5, RRE3 and RRE25 (member of β-subclass) and
Rhizobium leguminosarum bv. phaseoli RRE6 and R.
undicola RRE36 (member of α-subclass) were capable of
establishing as endophytes of maize plant. In the presence
of nitrate, the plant growth promotion effect produced by
endophytes (RRE6 and RRE5) was more enhanced; RRE6
showed better effect as compared to RRE5. From this study
it can be suggested that one of the sites of infection by
endophytic bacteria is root of the host plant. The
endophytic behavior and colonization ability of bacterial
endophytes (RRE5 and RRE6) in the maize plants can be
enhanced by nitrate supplementation.

INTRODUCTION
Plant nutrition is one of the most important factors that
increase plant productivity. Nitrogen (N) is the most
recognized in plants for its presence in the structure of the
protein molecule. In addition, N is found in such important
molecules as purines, pyrimidines, porphyrines, and
coenzymes. Purines and pyrimidines are found in the
nucleic acids RNA and DNA, which are essential for
protein synthesis. The porphyrin structure is found in such
metabolically important compounds as the chlorophyll
pigments and the cytochromes, which are essential in
photosynthesis and respiration. Coenzymes are essential to
the function of many enzymes. Accordingly, nitrogen plays
an important role in the synthesis of the plant constituents
through the action of different enzymes (Jones et al. 1991).
Nitrogen limiting conditions increase volatile oil
production in annual herbs. Nitrogen fertilization has been
reported to reduce essential oil content in creeping juniper
(Juniperus horizontalis) (Robert 1986), although it has
been reported to increase total essential oil yield in thyme

(Thymus vulgaris L.) (Baranauskienne et al. 2003). Munsi
1992 indicated that for improvement in production of
essential oil from a crop like Japanese mint (Mentha
arvensis L), a judicious application of nitrogen is required.
Each increase in N level increased the dill seed (Anethum
graveolens L.) up to 90 kg ha-1 but further increase did not
affect the seed yield significantly (Randhawa et al. 1996).
Nitrogen fertilization increased the vegetative growth,
essential oil, fixed oil, total carbohydrates, soluble sugars
and NPK content of some Apiaceae (Anis, coriander and
sweet fennel) and Nigella sativa L. plants (Khalid 1996;
Khalid 2001). Zheljazkov and Margina (1996) established
that plant height, branching, and essential oil content of
mint (Mentha piperita and Mentha arvensis) increased with
increasing N fertilizer rates. However, plant vegetative
growth was not significantly affected by the increase of N
fertilizer rates. With the increased N fertilizer rates, the
fresh herbage yield from the first cut increased by 13 to
72%, and that from the second cut by 23 to 78% compared
to the control. Nitrogen fertilization has been shown not
only to improve vegetative growth, but also to alter the

essential oil yield and composition of Japanese mint
(Mentha arvensis) (Saxena and Singh 1998). The influence
of N fertilizers on the yield of crop, as well as on the
production and composition of the essential oil and some
other chemical characteristics of thyme was investigated by
Baranauskienė et al. 2003. It was found that N fertilizer
increased thyme crop, but differences in the yield of
essential oil were not remarkable. However, the use of
certain amounts of nitrogen fertilizers resulted in higher
yields of essential oil obtainable from the cultivation area
unit (dm3 ha-1). The effects of N fertilizer on yield and
quality of basil (Ocimum basilicum L.) were investigated
by Arabaci and Bayram (2004) and they found that
intensive N fertilization increased the amount of green herb
yield, essential oil concentration and essential oil yield.
According to Ashraf et al. (2006), a field experiment was
conducted to study the effect of N fertilization level on the
content and composition of oil, essential oil and minerals in
black cumin (Nigella sativa L.) seeds. Sixty-three-day-old
plants were supplied with varying levels of N, i.e., 0, 30,
60, and 90 kg N ha−1. The fixed oil content of the seeds
ranged from 32.7% to 37.8% and it remained almost
unchanged at the two higher external N regimes, i.e., 60
and 90 kg N ha−1, but at 30 kg N ha−1 the oil content
increased significantly; Increasing N rate did not affect the
content of nutrient content in the cumin seeds. Akbarinia et
al. (2007) indicated that with increasing of N to 60 kg ha-1,
there was a significant increase in coriander (Coriandrum
sativum L.) seed yield, but the highest essential oil and
fatty acids content were obtained with 90 kg N ha-1. Senthil
Kumar et al. (2009) revealed that application of N at 93.75
kg ha-1 gave the highest plant height, number of laterals,
fresh and dry weight of shoot, dry matter production, fresh
herbage yield and essential oil yield of Davana (Artemisia
pallens Wall.). Nitrogen fertilization had different effects
on mint chemotypes, with M. x piperita, linalool
chemotype, being the only genetic material where nitrogen
fertilization resulted in higher total dry mass (Luciana et al.
2010). Hellal et al. (2011) indicated that applying N
fertilizer increased the growth, yield and chemical
constituents of dill (Anethum graveolens L.) plant
compared to the untreated control, the highest values of

vegetative growth, oil yield and NPK content were
recorded by the treatment of 100 kg N ha -1.
Anise (Pimpinella anisum L., Apiaceae) has been used
as an aromatic herb and spice since Egyptian times and
antiquity and has been cultivated throughout Europe
(Hänsel et al. 1999). In folk medicine, anise is used as an
appetizer, tranquillizer and diuretic drug (Tyler et al. 1988;
Lawless 1999). The traditional use of Pernod, Ouzo,
Anisette, Raki, and many other anise-flavoured drinks after
a heavy meal is a familiar example of its antispasmodic
effect, especially in the digestive tract (Hänsel et al. 1999).
Dried ripe fruits of anise, commercially called aniseeds
(Anisi fructus), contain the whole dry cremocarp of anise
(P. anisum). For medical purposes, they are used to treat
dyspeptic complaints and catarrh of the respiratory tract,
and as mild expectorants. It was also reported that extracts
from anise fruits have therapeutic effects on several
conditions, such as gynaecological and neurological
disorders (Czygan and Anis 1992; Lawless 1999).
Ethanolic extract of anise-fruits contains trans-anethole,
methylchavicol (estragole), eugenol, psedoisoeugenol,
anisaldehyde, coumarins (umbelliferon, scopoletin), caffeic
acid derivatives (chlorogenic acid), flavonoids, fatty oil,
proteins, minerals, polyenes and polyacetylenes as its
major compounds (Hänsel et al. 1999).
Coriander (Coriandrum sativum L.) is a culinary and
medicinal plant and belongs to the Apiaceae family. This
plant has economic importance since it has been used as
flavoring agent in food products, perfumes and cosmetics.
As a medicinal plant, C. sativum L. has been credited with
a long list of medicinal uses. Powdered seeds or dry
extract, tea, tincture, decoction or infusion have been
recommended for dyspeptic complaints, loss of appetite,
convulsion, insomnia and anxiety (Emamghoreishi et al.
2001). Moreover, the essential oils and various extracts
from coriander have been shown to possess antibacterial
(Burt 2004), antioxidant (Wangensteen et al. 2004),
anticancerous and antimutagenic (Chithra and Leelamma
2004) activities. Many phytochemical studies so far
investigated the chemical composition of the essential oil
from C. sativum L. fruits from different origins (Steinegger
and Hänsel 1988). Evaluations of the essential oil

composition extracted from leaves have also been reported
(Eyres et al. 2005). The coriander (Coriandrum sativum L.)
fruit essential oil yields showed marked increase during
maturation process and linalool was the main compound at
the fruiting stage (Kamel et al. 1994).
Due to their unique and preferred flavor and aroma, the
swollen bases of sweet fennel (Foeniculum vulgare var.
dulce, Apiaceae) are freshly consumed in salads or cooked
as a kitchen vegetable. The major constituents of fennel
essential oil such as anethole and limonene are also used as
essence in cosmetics and perfumes and for some medicinal
purposes (Marotti et al. 1993; Stuart 1982).
Sandy soils generally have fine grained texture. They
retain very little water, fertilizers or nutrients which means
they are extremely poor. They are prone to over-draining
and summer dehydration, and in wet weather can have
problems retaining moisture and nutrients and can only be
revitalized by the addition of organic matter. Sandy soils
are light and easy to dig, hoe and weed. In addition, arid
regions in Egypt are characterized by low nutrient contents
(especially N) which negatively affect growth and
productivity of medicinal and aromatic plants including
anise, coriander and sweet fennel (Abd-Allah et al. 2001).
The main objective of the present investigation was to
study the effect of different levels of N fertilizers on the
morphological and biochemical contents of anise, coriander
and sweet fennel plants under arid regions conditions.

MATERIALS AND METHODS
Experiments were carried out in the arid region at the
Experimental Farm of the Desert Development Center
(DDC) in Sadat City, American University, Egypt, during
two successive seasons, 1992/93 and /1993/94. The area of
DDC had been recently reclaimed and had not been
cultivated before. Physical and chemical properties of the
Typic Torrifluvents soil (USDA 1999) used in this study
(0-50 cm depth) were determined according to Jackson
(1973) and Cottenie et al. (1982) and are presented in Table
1. Seeds of coriander and anise were provided by the
Department of Medicinal and Aromatic Plants, Ministry of
Agriculture, Giza, Egypt as follows: fertilizers were added
to all plots as follows: cattle manure (50 m3 ha-1),
phosphorus (500 kg ha-1) as calcium super phosphate
(15.5% P2O5) and potassium (375 kg ha-1) as potassium
sulphate (48% K2O); whereas sweet fennel seeds were
imported from France. Sweet fennel seeds were sown in the
third week of October during both seasons. The seedlings
of sweet fennel were transplanted into the open field 45
days after sowing. At the same time, the seeds of coriander
and anise were sown directly in the open field. The
experimental design was a complete randomized block
with four replicates. The experimental area (plot) was 30
mÂ˛ (4 m x 7.5m) containing 15 rows; the distance between
hills was 25 cm and 50 cm between the rows. Thinning for
two plants per hill was made 45 days after cultivating the
plants in the open field. The sprinkler irrigation system was
used in this experiment. All agriculture practices other than
experimental treatments were performed according to the

17

recommendations of the Ministry of Agriculture, Egypt.
Plots were divided into four groups subjected to N
application to soil as ammonium sulphate [(NH4)2SO4]
(20% N) with the rates of 0, 100, 150 and 200 kg ha-1.
Table 1. Mechanical and chemical analyses of the Typic
Torrifluvents soil
Sand

Silt
%
13.0

79.7
Ca++

Mg++

Na++

4.9

5.6

1.9

K
mg g -1
0.5

Clay Gravel
7.3

18.7

pH

ECe (dS m-1)

8.7

2.0

K+
CO3 HCO3mg g -1
0.6
0.8
1.9
Fe

Cu

5400

400

Zn
ug mg-1
300

Cl -

SO4--

18.6

0.2
Mn
1600

Harvesting
At fruiting stage, the plants were harvested during the
two seasons. Vegetative growth characters measurements
[Plant height (cm), Leaf number (plant-1), Branch number
(plant-1), Umbel number (plant-1), Herb fresh weight (g
plant-1), Herb dry weight (g plant-1) and Fruit yield (g plant1
)] were recorded.
Essential oil isolation
Ripening fruits were collected from each treatment
during the first and second season, and then 100 g from
each replicate of all treatments was subjected to hydrodistillation for 3 h using a Clevenger type apparatus
(Clevenger 1928). The essential oil content was calculated
as a weight/volume percentage. In addition, total essential
oil yield (g plant-1) was calculated by using the dry weight
of the fruits.
Total carbohydrates and soluble sugars
Total carbohydrates (TC) and soluble sugars
concentrations in leaves (collected at the end of the first
and second season of each treatment) were determined
according to Ciha and Brun (1978) with some
modifications. Samples of 100 mg were homogenized with
10 mL of extracting solution [glacial acetic acid: methanol:
water, 1:4:5, v/v/v for soluble sugars or glacial acetic acid:
H2SO4 (1N): water, 1:4:5, v/v/v for TC]. The homogenate
was centrifuged for 10 min at 3,000 rpm and the
supernatant was decanted. The residue was resuspended in
10 mL of extracting solution and centrifuged another 5 min
at 3,000 rpm. The supernatant was decanted, combined
with the original extract and made up to 50 mL with water.
For measurement of total carbohydrates and soluble sugars,
a phenol-sulfuric acid assay was used (Dubois et al. 1956).
A volume of 0.5 mL of 5% (v/v) phenol solution and 2.5
mL of concentrated sulfuric acid were added to 0.5 mL
aliquots. The mixture was shaken, heated in a boiling
water-bath for 20 min and cooled to room temperature. The
absorption was then determined by spectrophotometry at
490 nm.

18

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5 (1): 15-21, May 2013

Fixed oil, nutrients and crude protein determination
Fixed oil Extraction: Fifty grams of fruits were crushed
to coarse powder and extracted with petroleum ether (40-60
Îż
C) in a Soxhlet apparatus (AOAC 1970). N, protein, P and
K (in the leaves) of both seasons of each treatment were
determined using the methods described by the AOAC
(1970) as follows:
The washed and dried materials were ground to fine
powder with mortar and pestle and used for dry ashing.
For analysis of K, the powdered plant material (0.2 g)
was taken in precleaned and constantly weighed silica
crucible and heated in muffle furnace at 400 0C till there
was no evolution of smoke. The crucible was cooled in
desicator at room temperature. The ash, totally free from
carbon was moistened with conc. H2SO4 and heated on hot
plate till fumes of sulphuric acid was evolved from the
silica crucible, with sulphated ash was again heated at 600
0
C in muffle furnace till weight of sample was constant (34 hrs). One gram sulphated ash was dissolved in 100 ml 5
% conc. HCl in a beaker to obtain solution for
determination of K through flame photometry. Standard
solution of each mineral was prepared and calibration curve
drawn for K element using flame photometry (Alexander
1963).
For the determination of protein and nitrogen using
Micro Kjeldahl method, 1 g of plant sample taken in a
Pyrex digestion tube and 30 ml of conc. H2SO4 carefully
added, then 10 g potassium sulphate and 14 gm of copper
sulphate, mixture was placed on sand both on a low flame
just to boil the solution. It was further heated till the
solution became colorless and clear, allowed to cool,
diluted with distilled water and transferred to 800 ml
Kjeldahl flask, washing the digestion flask, three or four
pieces of granulated zinc and 100 ml of 40 % caustic soda
were added and the flask was connected with the splash
heads of the distillation apparatus. Next 25 ml of 0.1 N
sulphuric acids was taken in the receiving flask and
distilled; it was tested for completion of reaction. The flask
was removed and titrated against 0.1 N caustic soda
solution using Methyl Red indicator for determination of
nitrogen, which can be calculated to give the protein
content (Lynch and Barbano 1999).
For determination of phosphorous, a 2 g sample of plant
material was brought into a 100 ml conical flask, two
spoons of Darco-G-60 were added followed by 50 ml of
0.5 M NaHCO3 solution. Next, the flask was corked and
shaked for 30 min on a shaker. The content was filtered and
the filtrate was collected in a flask from which 5 ml filtrate
were taken to a 25 ml volumetric flask. To this solution 2
drops of 2, 4- paranitrophenol and 5 N H2SO4 drops were
added with intermittent shaking till the yellow color
disappeared. The content was diluted to 20 ml with distilled
water and then 4 ml ascorbic acid were added. Then the
mixture was well shaken and the intensity of blue color at 660
nm on colorimeter was measured and compared to phosphorus
standards to obtain P concentrations (King 1932).
Statistical analysis
In this experiment, one unique factor was considered:
nitrogen application as ammonium sulphate (0, 100, 150

and 200 kg ha-1 ). For each treatment there were four
replicates. The experimental design followed a complete
random block design. The averages of data for both seasons
were statistically analyzed using one way analysis of
variance (ANOVA-1) using the STAT-ITCF program
(Foucart 1982). The least of significant differences between
means was calculated using least significant difference
(LSD) at 5% according to Snedecor and Cochran (1990).

RESULTS AND DISCUSSION
Effect of N fertilization on plant growth and
development
Data in Table 2 shows the response of anise, coriander
and sweet fennel plants to the different rates of N. All
Nitrogen treatments produced significantly higher values
than the control and significantly improved plant growth
characters [plant height (cm), leaf number (plant-1), branch
number (plant-1), umbel number (plant-1), herb fresh weight
(g plant-1), herb dry weight (g plant-1) and fruit yield (g
plant-1)]. Their highest values were recorded when plants
were treated with 200 kg N ha-1. The highest values of
plant growth characters (respectively) were 44.5, 32.6, 8.5,
29.7, 19.8, 9.4 and 5.9 for anise; 80.0, 66.0, 7.1, 29.9, 15.9,
12.3 and 5.9, for coriander; 98.9, 21.3, 5.3, 10.4, 118.4,
90.4 and 21.3, for sweet fennel. The ANOVA indicated
that the increases in vegetative growth characters were
significant for N treatments (Table 2). The positive effects
of N fertilization may be due to the important physiological
role of N in molecule structure as porphyrin. The porphyrin
structure is found in such metabolically important
compounds as the chlorophyll pigments and the
cytochromes, which are essential in photosynthesis and
respiration. Coenzymes are essential to the function of
many enzymes. Accordingly, nitrogen plays an important
role in synthesis of the plant constituents through the action
of different enzymes activities and protein synthesis (Jones
et al. 1991) that reflected in the increase in growth
parameters of plants such as anise, coriander and sweet
fennel plants. Also, these results are in accordance with
those obtained by Khalid (1996, 2001) on some Apiaceae
and Nigella sativa L. plants; Ashraf et al. (2006) on cumin;
Akbarinia et al. (2007) on coriander; Hellal et al. (2011) on
dill (Anethum graveolens L.), all of whom reported that N
fertilizer treatments were superior to the control treatment
and significantly improved the vegetative growth
characters of family Apiaceae.
Effect of N fertilization on the essential oil content
The effects of different treatments of N on the essential
oil content (% or ml plant-1) extracted from anise, coriander
and sweet fennel fruits are represented in Table 3.
Generally, all levels of N as soil application progressively
increased the essential oil of anise, coriander and sweet
fennel plants compared to the control. The last level of N
(200 kg N ha-1) seemed to be optimal for obtaining a higher
concentration of essential oil than the control and other
treatments: 0.6, 0.1 and 0.7% more than the control for
anise, coriander and sweet fennel, respectively. The

ANOVA indicated that the increase in
essential oil (%) was significant (P<0.05) in
anise and not significant in coriander and
sweet fennel. The increase in essential oil
yield (g plant â&#x2C6;&#x2019;1) were not significant in
anise and coriander but was significant in
sweet fennel (Table 3). The effect of
different N treatments on essential oil may
be due to its effect on enzyme activity and
metabolism of essential oil production in
peppermint plant (Burbott and Loomis
1969). These results were in accordance
with those obtained by Khalid (1996 and
2001) on some Apiaceae and Nigella sativa
L. plants; Ashraf et al. (2006) on cumin;
Akbarinia et al. (2007) on coriander; Hellal
et al. (2011) on dill (Anethum graveolens
L.), who reported that N has a positive
effect on the quantity of essential oil
extracted from Apiaceae plants.
Effect of N fertilization on the fixed oil
content
Data presented in Table 3 shows that the
intensive N fertilization produced an
increase in the accumulation of fixed oil (%
or ml plant-1) extracted from anise,
coriander and sweet fennel fruits, with the
highest content of fixed oil obtained with
the highest N dose of 200 kg ha-1. Fixed oil
contents were 8.1, 2.7 and 0.4% higher than
the control for anise, coriander and sweet
fennel, respectively. The ANOVA indicated
that the increase in fixed oil (%) of anise,
coriander and sweet fennel was significant.
The increase in fixed oil yield (ml plant-1)
was significant for anise and coriander while
not significant for coriander. These results
were similar to those of Khalid (1996) on
some Apiacea plants; Khalid (2001) a
Ashraf et al. (2006) on Nigella sativa L.;
and Akbarinia et al. (2007) on coriander
(Coriandrum sativum L.)

Effect of N fertilization on the total
carbohydrates and soluble sugars
contents
Table 3 shows that total content of
carbohydrates and soluble sugars in anise,
coriander and sweet fennel increased with
increasing N rates. The highest contents of
total carbohydrates and soluble sugars were
recorded when plants were treated with 200
kg ha-1 compared with the control or other
treatments. Total carbohydrates were 0.6%,
7.1% and 0.4% higher than the control and total soluble
sugars, 0.8%, 3.2%, and 0.4%) for anise, coriander and
sweet fennel, respectively. The ANOVA indicated that the
increase in total carbohydrates in anise and coriander was
significant, while it was not significant for sweet fennel.

N
P
K
(%) (%) (%)

2.3
2.6
2.7
2.9

0.7
0.8
0.9
1.0

1.5
1.6
1.9
2.0

0.3 NS NS
1.6
1.8
1.9
2.8

0.3
0.4
0.5
0.5

2.9
3.1
3.2
3.3

0.1 NS NS
1.8
1.9
2.0
2.1

0.6
0.7
0.8
0.9

2.1
2.3
2.5
2.6

NS NS 0.1

The increase in soluble sugars in anise and sweet fennel
was not significant, but it was significant in coriander.
These results may be due to the increase in chlorophyll
content, and consequently, photosynthesis efficiency,
induced by N. So it showed that total carbohydrates and

Â

20

5 (1): 15-21, May 2013

soluble sugars contents increased with application of N
(Jones et al. 1991).
Effect of N fertilization on the total protein content
Protein content was positively affected by soil
application of N (Table 3). The treatment of 200 kg ha-1
resulted in the highest protein content in anise, coriander
and sweet fennel plants: 2.4%, 3.9% and 0.4% higher than
the control, respectively. The ANOVA indicated that the
increase in total protein content was significant for anise
and coriander while not significant for sweet fennel. These
results may be due to the influence of N on the ribosome
structure and the biosynthesis of some hormones
(gibberellines, auxins, cytokinins) involved in protein
synthesis (Jones et al. 1991; El-Wahab and Mohamed
2007).
Effect of N fertilization on the mineral content
It is evident from the Table 3 that NPK content
gradually increased in all treatments as compared with the
control treatment. With respect to the effect of the N levels,
data indicated that applying 200 kg ha-1 brought about the
highest values of NPK content in anise, coriander and
sweet fennel plants: 0.6%, 0.3% and 0.5% for anise; 1.2%,
0.2 % and 0.4% for coriander; 0.3%, 0.3%, and 0.5% for
sweet fennel higher N, P and K than the control
respectively. The ANOVA indicated that the increase in N
content of anise and coriander was significant while it was
not significant for sweet fennel. The increase in P content
of anise, coriander and sweet fennel was not significant.
The increase in K content of anise and coriander was not
significant, but the increase in sweet fennel was. The
increase in the essential minerals according to the N
treatments may be due to the increase in the dry matter of
plant materials (El-Wahab and Mohamed 2007).

CONCLUSION
It may be concluded that N treatments resulted in
positive increase in plant growth characters, and content of
essential oil, fixed oil, total carbohydrates, soluble sugars,
protein, and nutrients. The highest values were recorded
when plants were treated with 200 kg ha-1 for anise,
coriander and sweet fennel.

Abstract. Hendawy SF, Hussein MS, Youssef AA, El-Mergawi RA. 2013. Response of Silybum marianum plant to irrigation intervals
combined with fertilization. Nusantara Bioscience 5: 22-29. This study was investigated to evaluate the influence of different kinds of
organic and bio fertilization under different irrigation intervals on the growth, production and chemical constituents of Sylibium
marianum plant. Data indicated that all studied growth and yield characters were significantly affected by the duration of irrigation
intervals. Fertilizer treatments had a primitive effect on growth and yield characters. The interaction between irrigation intervals and
fertilizer treatments has a clear considerable effect on growth and yield characters. The obtained results indicated the favorable effect of
organic and bio fertilizers which reduce the harmful effect of water stress. Different treatments had a pronounced effect on silymarin content.
Key words: Sylibium marianum, silymarin, bio fertilization and irrigation intervals.

INTRODUCTION
Milk thistle (Silybum marianum L. Gaertn.), a member
of the Mediterranean Basin, as a crop and weed on
agricultural plantations, it occurs in many European
countries, North Africa, South and North America, Central
and Western Asia and southern Australia (Carrier et al.
2002).The pharmaceutical compound of milk thistle is
derived from its fruits, which are achenes (Fructus silybi
mariani). In their dry pericarp and seed coat the plant
accumulates a group of flavonolignans commonly referred
to as silymarin (Cappelletti and Caniato 1984). Taxifolin is
their precursor. The main flavonolignans of milk thistle are
silybinin, isosilybinin, silydianin and silychristin. Several
other compounds of that type have also been identified, but
their importance in the silymarin complex is insignificant
(Kurkin et al. 2001). Silymarin, derived from the seeds of
milk thistle plant has been used widely for the treatment of
toxic liver damage (Dewick 1998). Silymarin primarily
consists of an isomeric mixture of six phenolic compounds:

silydianin, silychristin, diastereoisomers of silybin (silybin
A and B), and diastereoisomers of isosilybin (isosilybin A
and B) (Lee et al. 2007).
The compost must be added to conventional NPK
fertilizer to improve soil structure, making the soil easier to
cultivate, encouraging root development, providing plant
nutrients and enabling their increased uptake by plants.
Moreover, compost aids water absorption and retention by
the soil, reducing erosion and run-off and thereby
protecting surface waters from sedimentation, help binding
agricultural chemicals, keeping them out of water ways and
protecting ground water from contamination (leaMaster et
al. 1998). Compost has already been established as a
recommended fertilizer for improving the productivity of
several medicinal and aromatic plants, as peppermint
(O’Brien and Barker 1996), Tagetes erecta (Khalil et al.
2002), Sideritis montana (El-Sherbeny et al. 2005), Ruta
graveolens (Naguib et al. 2007) and Dracocephalum
moldavica L. ( Amer 2008). Compost tea is a highly
concentrated microbial solution produced by extracting

Damghani 2001). It appears that the effect
of water stress on economic yields of
medicinal plants which are mainly
secondary metabolites, are somehow
positive (Baher et al. 2002). In many cases,
a moderate stress could enhance the content
of secondary metabolites.
This current experiment targeted the
evaluation of the influence of different kinds
of organic and bio fertilization under
different irrigation intervals on the growth,
production and chemical constituents of
Sylibium marianum plant.

MATERIALS AND METHODS
Field experiment
Location
The field experiment was carried out at
El-Nubareia Research Station (El-Behira
Governorate, Egypt), National Research
Centre, to investigate the influence of
Chemical, organic and bio fertilizers under
different irrigation intervals on growth,
yield and chemical constituents of milk
thistle.

Figure 1. Inflorescense of milk thistle (Silybum marianum L. Gaertn.)

beneficial microbes from vermicompost and or compost.
Compost tea provides direct nutrition as a source of foliar
and soil organic nutrient and as chelated micronutrients for
easy plant absorption. Also, compost tea provide microbial
functions, that compete with disease causing microbes,
degrade toxic pesticides, produce plant growth hormones,
mineralize plant available nutrients and fix nitrogen
(Hendawy 2008).
Arbuscular mycorrhiza (AM) fungi (Endogonaceae)
form a mutualistic relationship with the roots of most plant
species. This plant-fungus association involves the
translocation of carbon from the plant to the fungus and
enhanced uptake and transport of soil nutrients, primarily
phosphorus, to the plant via the fungus (Newman and
Reddel 1987). Other potential benefits of AM fungal
colonization to host plants include improved uptake of
poorly mobile nutrients such as zinc (Gildon and Tinker
1983), improved plant water relations (Allen and Allen
1986) and reduced pathogenic infections (Newsham, et al.
1995). AMF can also benefit plants by stimulating the
production of growth regulating substances, increasing
photosynthesis, improving osmotic adjustment under
drought and salinity stresses and increasing resistance to
pests and soil borne diseases (Al-Karaki 2006).
However, water deficit is a limiting factor in
production of many field crops (Kafi and Mahdavi
Damghani 2001; Munns 2002) and water stress causes
different morphological, physiological and biochemical
changes including: leaf area reduction, leaf senescence and
reduction in cell development (Kafi and Mahdavi
Damghani 2001), stomatal closure (Safar-Nezhad 2003)
and photosynthetic limitation (Kafi and Mahdavi

The seeds were directly sown in 20th of October 2010.
Each plot was 13.5 m2 consisting of 9 rows with a distance
of 50 cm between the rows and 30 cm between each
successive plant.. Weeding and thinning was done after 30
days of plantation. Recommended agronomic practices
were adopted.
Super phosphate or compost was added during
preparing soil. The other chemical fertilizers (Ammonium
nitrate and Potassium sulphate were divided into two equal
portions during the growing season, the 1st portion was
added after one month of sowing, while the second one was
applied after one month from the 1st. Tea compost (Table 2,
3) was sprayed after 60 days from sowing and repeated
after 15 days.Vesicular arbscular mycorrhiza (VAM) fungi
which contained 3 effective strains representing Glomus
etunicatum, Glomus fasciculatum and Glomus intraradices.
VAM fungi was used for soil inoculation. The VAM
inoculation was applied into sowing hills at a rate of 5
mL/hill. The amount contained about 200 VAM spores/hill.
The effect of the above treatments was measured by
plant height, branches number, capitula number/plant, seed
yield and silymarin content.
Table 2. Microbial population of organic compost tea
Constituent

RESULTS AND DISCUSSION
Vegetative growth and yield
Irrigation intervals
Data tabulated in Table 4 indicated that all studied
growth and yield characters were significantly affected by
the duration of irrigation intervals.
By increasing the severity and duration of drought from
3 days to 9 days, plant height (cm) showed significant
reduction. Such reduction in plant height in response to
drought may be due to blocking up of xylem and phloem
vessels thus hindering any translocation through (Lovisolo
and Schuber 1998). Similar results were obtained by Singh
et al. (2006) and Khalil et al. (2010).

Data on hand, illustrated also that, number of
branches/plant increased significantly with decreasing of
irrigation, this may be due to that drought reduced cyclingdependent kinase activity results in slower cell division as
well as inhibition of growth (Schuppler et al. 1998). This
supported by the results of (Rahmani et al. 2008) on
Calendula officinalis L. and (Taheri et al. 2008) on
Cichorium intybw L.
Significant higher numbers of flowers head/plant and
seed yield (g/plant) were recorded with the shortest
irrigation interval (3 days) followed by (6 days). The
decrease in yield attributes under the longest irrigation
interval (9 days) may be due that water stress changing the
hormonal balance of mature leaves, thus enhancing leaf
senescence and hence the number of active leaves
decreased, as well as leaf area was reduced by water
shortage, which was attributed to its effect on cell division
and lamina expansion. When the number of active leaves
decreased the light attraction and CO2 diffusion inside the
leaf decreased and the total capacity of photosynthesis
decreased, therefore, the photosynthetic materials that
transferred to seeds will decreased (Ahmed and Mahmoud
2010; Moussavi et al. 2011).
Fertilizer treatments
Data tabulated in Table 5 show that fertilizer
treatments had a significant effect on growth and yield
characters of Silybum marianum plants. The mean values
of plant height were 174.33, 164.33, 168.33, 171.0 and
179.67 cm as a result of NPK, compost, compost+
mycorrhiza, compost+compost tea and compost+compost
tea+mycorrhiza treatments, respectively. So, the highest
value of plant height was obtained as a result of
compost+compost tea+mycorrhiza treatment.
Table 5. Effect of fertilizer treatment on vegetative growth and
yield of Silybum marianum
Seed
yield
(g/plant)
18.15
15.74
18.90
16.49
19.37

The results in Table 5 reveal that, fertilizer treatments
had a pronounced effect on branches number. It can be
noticed that, mean values of branches number recorded
8.33, 6.33, 7.33, 7.33 and 8.00/plant were obtained from
NPK, Compost, Compost+mycorrhiza, compost+compost
tea and compost+compost tea+mycorrhiza treatments,
respectively. Thus, the maximum mean value of branches
number/plant (8.33) was obtained as a result of NPK
treatment followed by compost+compost tea+mycorrhiza
treatment, which recorded 8.00/plant. There is no
significant difference between NPK treatment and
compost+compost tea+mycorrhiza treatment.
The averages of heads flowers number were 21.33,
17.00, 16.67, 17.00 and 19.00/plant as a result of NPK,
Compost, Compost+mycorrhiza, compost+compost tea and
compost+compost tea+mycorrhiza treatments, respectively.
Thus, the maximum mean value of flowers heads
number/plant (21.33) was obtained from NPK treatment
followed by compost+compost tea treatment, which
recorded 19.00/plant.
It is evident from data in Table 5 that fertilizer
treatments had a significant effect on seed yield (g/plant).In
this respect, mean values of seed yield (g/plant) were
18.15, 15.74, 18.90, 16.49 and 19.37 g/plant as a result of
as a result of NPK, compost, compost+mycorrhiza,
compost+compost tea and compost+compost tea+
mycorrhiza treatments, respectively. Therefore, compost+
compost tea+mycorrhiza treatment gave the highest mean
value of seed yield (19.37g/plant) followed by
compost+mycorrhiza treatment which recorded (18.90
g/plant).
The promotion effect of compost on the growth and
yield of plant could be explained through the role of
organic materials including composts in improving soil P
availability (Gichangi et al. 2009). Since during
composting, labile nutrients are converted into stabilized
organic material (Zucconi and De Bertoldi 1987), therefore
a large proportion of nutrients are labile. Composts provide
microbes not only with P but also C and N and are
therefore likely to induce changes in P pools that differ
from those of inorganic P addition (Hassan et al. 2012).
The favorable effects of the combination between
compost +compost tea+mycrohiza may be explained based
on the beneficial effects of them on the improvement soil
physical and biological properties and also, the chemical
characteristics resulting in more release of available
nutrient elements to be absorbed by plant root and its effect
on the physiological processes such as photosynthesis
activity as well as the utilization of carbohydrates. A
similar suggestion was made by Hanafy et al. (2002) on
rocket plants. Furthermore, this stimulative effect may be
related to the good equilibrium of nutrients and water in the
root medium (Abdelaziz and Balbaa 2007) or to the
beneficial effects of mycorrhiza on vital enzymes and
hormonal, stimulating effects on plant growth and yield.
Interaction treatments
The interaction between irrigation intervals and
fertilizer treatments has a clear considerable effect on
growth and yield characters (Table 6). It can be observed

26

5 (1): 22-29, May 2013

that the maximum mean value of plant height (190.00 cm)
was obtained from the combination treatment between
irrigation intervals every 3 days and fertilized with
compost+compost tea+mycorrhiza. On the other hand, the
lowest average of plant height (158.00 cm) was obtained
from the combination between irrigation intervals every 9
days and compost treatment. The variation in plant height
between maximum and the minimum values reached to
20.25%.
For branches number/plant, it can be observed that, the
highest mean value of branches number/plant (10.00/plant)
against the lowest value (5.00/plant) were obtained as a
result of the combination between irrigation intervals every
9 days and NPK treatment and the combination irrigation
intervals every 3 days with compost treatment,
respectively. The variation in branches number/plant
between maximum and the minimum values reached to
100%.
Data shown in Table 6 indicated that, the combination
between irrigation intervals every 3 days and NPK
treatment gave the highest mean value of flowers heads
number (25.00/plant),while the combination between
irrigation intervals every 9 days and compost+compost tea

treatment gave the lowest mean value (13.00/plant). The
variation in flowers heads number/plant between maximum
and the minimum values reached to 92.31%.
Concerning the interaction treatments, it can be noticed
that the combination between irrigation intervals every 3
days and compost+compost tea+mycorrhiza treatment
resulted in the maximum mean value of seed yield (23.40
g/plant) while the interaction between irrigation intervals
every 9 days and compost+compost tea treatment gave the
lowest one (13.00 g/plant). The variation in seed yield
(g/plant) between maximum and the minimum values
reached to 78.49%.
The obtained results indicated the favorable effect of
organic and bio fertilizers which reduce the harmful effect
of water stress through their effect on improving the soil
texture. The structural improvement can encourage the
plant to have a good root development by improving the
aeration in the soil. The favorable effects of these fertilizers
may be due to the role of organic material for continues
supply of nutrients, which improve some physical
properties of soil and increase water retention (AbdElmoez et al. 1995; Fliessbach et al. 2000).

Silymarin content
Data tabulated in Tables 7, 8 and 9 indicated that total
silymarin content (mg/g seed) ranged from 64.86 to 96.29
mg/g. The main constituent of silymarin were Silybin B
(17.70-26.54 mg/g) followed by Silychristin (17.48-24.93
mg/g). In this connection, dried extracts of milk thistle
seeds contain approximately 60% silymarin, where
silymarin consists of four flavonolignans of silybinin (~ 50
to 60%), isosilybinin (~ 5%), silychristin (~ 20%) and
silydianin (~ 10%) (Burgess, 2003). (Ibrahim et al. 2007)
found that the concentration and total yield of six silymarin
compounds showed wide variations between lines, varieties
and generations ranged from 11.92 to 62.85 mg/g seed and
between 329.8 to 2121.3 mg/plant, respectively. Six
silymarin compounds: silychristin, silydinin, silybin A,
silybin B, isosilybin A and isosilybin B were detected in
the extract of all tested treatments. These results were in
agreement with (Ibrahim et al. 2007).
Irrigation intervals
Data tabulated in Table 7 show that, the mean values of
total Silymarin content (mg/g seed) were 68.83, 72.65 and
85.41 mg/g were obtained as a result of irrigation intervals
at 3, 6 and 9 days, respectively.
Silybin B followed by silychristin were the main
components of silymarin. The maximum mean values of
Silybin B (23.34 mg/g) and Silychristin (22.03 mg/g) were
observed as a result of irrigation intervals every 9 days.
Drought stress increases the secondary products
percentage of more medicinal and aromatic plants, because
in case of stress, more metabolites are produce in the plants
and substances prevent from oxidization in the cells, but
secondary products content reduce under drought stress,
because the interaction between the amount of the
secondary products percentage and mass production is
consider important as two components of the secondary
products content and by exerting stress, increases the
secondary products percentage but mass production
decreases by the drought stress, therefore secondary
products content reduces. The data from (de Abreu and

Irrigation
intervals
3 days

6 days

9 days

Mazzafera 2005) showed that also the total amount of some
secondary plant products per plant indeed is significantly
higher in plants grown under drought stress than in those
cultivated under normal conditions. Although stressed
plants had been quite smaller, the product of biomass and
substance concentration yields in a 10% higher amount of
phenolic compounds; however, the total content of
betulinic acid was nearly the same in plants when grown
under drought stress or under standard conditions. Also the
studies published by Nogues et al. (1998), who found a
massive increase of phenolic compounds in stressed peas,
allow calculating the overall yield of the related substances.
Despite the fact that the total biomass of pea plants grown
under drought stress is just about one third of those
cultivated under standard condition, the overall amount of
anthocyanins (product of biomass and anthocyanin
concentration) is about 25% higher in the stressed plants.
Apart from that, the overall yield of total flavanoids was
nearly the same in Pisum sativum plants grown under
drought stress or under non-stress conditions.
Fertilizer treatments
Data tabulated in Table 8 indicated the effect of
different fertilizer treatments on silymarin content (mg/g).
Total silymarin content ranged from 75.52 to 78.85 mg/g.
Compost+mycorrhiza treatment gave the maximum mean
values of total silymarin content (78.85 mg/g) followed by
Compost+compost tea+mycorrhiza treatment which gave
76.70 mg/g. The highest mean values of Silybin B (21.48
mg/g) and Silychristin (20.49 mg/g) were obtained as a
result of compost+mycorrhiza treatment compared with
other treatments.
As for the favorable effect of applying organic and/or
bio fertilizers on silymarin content may be due to effect of
these fertilizers on accelerating metabolism reactions as
well as stimulating enzymes. Application of bio fertilizers
and compost significantly improved secondary products
such as essential oil, rutin and coumarin (El-Sherbeny et al.
2007 a, b). Variations in plant growth and active principles
in mycorrhizae inoculated plants have been reported for

Â

28

5 (1): 22-29, May 2013

many other medicinal plants (Sailo and Bagyara 2005;
Copetta et. al. 2006).
Interaction treatments
It can be noticed that compost+ mycorrhiza treatment
under 9 days irrigation intervals gave the maximum value
of total silymarin content (96.29 mg/g) followed by
compost treatment under the same irrigation intervals
which gave 89.24 mg/g (Table 9). The lowest value of
Sylimarin content (64.86 mg/g) was obtained as a result of
compost+mycorrhiza treatment under 3 days irrigation
intervals.
Moreover, the highest values of Silybin B (26.54 mg/g)
and Silychristin (24.93 mg/g) were observed as a result of
compost+ mycorrhiza treatment under 9 days irrigation
intervals. In this respect, mycorrhiza fungi play a critical
role in interest cycling and ecosystem function. They
improve plant growth and survival through a mutuality
relationship in which photosynthates are exchanged for
increased access to water and nutrients (Kernaghan 2004).
These effects may be played an important role to increase
the secondary metabolites accumulation.

CONCLUSION
All presented data indicated that all studied growth and
yield characters were significantly affected by the duration
of irrigation intervals also organic and bio fertilizer showed
a primitive effect on growth and yield characters. The
interaction between irrigation intervals and fertilizer
treatments has a clear considerable effect on growth and
yield characters. Organic and bio fertilizers can reduce the
harmful effect of water stress.

INTRODUCTION
The wood properties vary as a result of variation in
fiber morphology within each annual ring formed, between
trees and between stands (Zobel and van Buijtenen 1989).
Wood quality characteristics can be influenced by both tree
growth condition and genetic factors (Jyske 2008; Gaspar
2009). Wood anatomical structure relates to wood product
properties like flexibility, plasticity, resistance, and optical
features (Panshin and Zeeuw 1980; Zhang 1997; StGermain and Krause 2008). Fiber length, lumen size and
cell wall thickness have influence on the rigidity and
strength properties (Oluwafemi and Sotannde 2007).
Plus tree selection is one of the first steps and used
method of obtaining material for forest tree improvement
programs (Zobel and Talbert 1984; Changtragoon 1996).
Plus trees are phenotypes judged but not proved by test to
be unusually superior in some quality and quantity, eg
growth rate, disirable growth habit, high wood density and

exeptional apparent resistance to disease and insect attack
(Nieuwenhuis 2000).
Regarding to wood economic importance and its usage
on human life and limitation of natural recourses,
determination of wood quality and appropriate application
for suitable usage is necessary. This is dependent on
identification of wood physical and anatomical properties
(Doosthosseini and Parsapajouh 1996) and finding the
relations between environmental and genetic factors on
them. Some studies carried out to determine wood fiber
properties of beech trees (Fagus orientalis Lipsky; Figure
1) and effect of them on strength properties (Akgul and
Tozluoglu 2009). They found that utilization of juvenile
woods on fiber production, can have contribution on raw
material supply. In previous studies were found the latitude
and altitude has major effects on variability in wood
properties within species and, could have impact on
juvenile wood rate production as well (Panshin and de
Zeeuw 1980).

Related studies can also provide knowledge and
guidance for Kiaei (2011) reported that altitude and height
of tree has effect on wood density and fiber biometry
properties of hornbeam-Carpinus betulus (L.). With
increase of altitude from sea level, the wood density, cell
wall thicknesses and rankle ratio were increased and the
fiber length, fiber diameter, fiber lumen diameter,
slenderness ratio and flexibility ratio values were
decreased. Varshoie et al. (2006) found that influence of
altitude on fiber thicknesses of beech trees is significant
however he did not find significant relation between
environmental factor and other biometry properties. On the
other study altitude did not affect on fiber length (Hosseini
2006). Ishiguri et al. (2007) suggested that the basic density
of core wood is a very important factor for the selection of
a plus tree in tree breeding for wood quality. Gaspar and et
al. (2009) studied the consequences of selection on wood
quality traits of Pinus Pinaster. They concluded that genetic
selection based on growth will not result in a decrease of
wood density, will not affect the occurrence of spiral grain,
and is possible to obtain an increase in the radial modulus
of elasticity. They suggest that selection for growth will
probably not affect negatively the wood properties at
future.
The wood of oriental beech is heavy, hard, strong and
highly resistant to shock. It is one of the most important
commercial woods in Iran. Oriental beech wood use as
particleboard, furniture, flooring veneer, mining poles

(props), railway tiles and paper (Kandemir and Kaya 2009).
In this study were investigated fiber and biometry
properties of beech wood and effect of altitude and
selection on them because we can identify application of
wood with knowledge about wood physical and anatomical
properties, and we can find a way for operating silviculture
programs for more genetic conservation and improvement
and extension of generation of suitable trees. This is
provided production plus quality wood in natural forest.

MATERIALS AND METHODS
Site study
Iranian beech forests are located on the northern slopes
of Alborz Mountains, Hyrcanian forests, within an altitude
of about 600-2000 m.a.s.l. They assemble a forest strip of
700 km length, located in three provinces of Guilan,
Mazandaran and Golestan (Salehi Shanjani et al. 2011).
This study was conducted at Shast Kalate forest, at the
Gorgan University of Agricultural Sciences and Natural
Resources on the Golestan province. It is located in
northern Iran (36° 41’ to 36° 45’ northern latitudes and 54°
20’ to 54° 24’ eastern longitudes) with an area of about
3716 ha and an altitude ranging from 100 to 1000 m above
sea level (Figure 2).This mixed deciduous forest is covered
different forest community such as Zelkova-Quercetum,
Parrotio-Carpinetum, Fageto-Carpinetum and Fagetum

Seth et al. 1997; Vahey et al. 2007). Some factors such as
soil, climate, and altitude and forest management lead to
appear differences on wood properties of timber of same
species (Doosthosseini and Parsapajouh 1997). The
hardwood plant species had significant difference on wood
density, fiber properties and mechanical strength (Kiaei and
Samariha 2011).
Even though a difference in altitude of about 300 m
could not seriously have effect fiber morphology and
biometry coefficients in beech trees, it seems that altitude
from sea level should play a positive role with beech trees
when the difference is greater than it (Hosseini 2006). StGermain and Krause (2008) found that latitude (along a
500 km transect) had no effect on tracheid length. Hosseini
(2006) observed altitude in the range of about 500 m no
important effect on beech fiber length.
In this investigation plus trees had long fiber, small
fiber diameter, wide fiber lumen and thin wall thickness.
The value of slenderness ratio and flexibility ratio in plus
trees was bigger than non-plus trees but the value of rankle
ratio in plus trees was lower than non-plus trees. The beech
plus trees are superior on phenotype in comparison with
beech non-plus trees, they have good stem form like stem
straightness, non-twisting bole, non-undulating, more clear
bole height (CBH), diameter polarity and crown polarity it
cause to increase fiber length and decrease fiber diameter.
The species with higher lengths, small diameter, thin wall
cell and large cell lumen are more desirable for paper
formation and strength (Monteoliva et al. 2005; Gaspar
2009). Regard to beech plus trees have been selected to
reach suitable industrial wood but results are shown that
non-plus trees in comparison with plus trees have more
desirable strength properties therefore they are can be used
on fiber board production and wood plus trees suitable for
fiber plate, rigid cardboard production.
Regard to stem form is one of easiest and quickest ways
to improve wood quality, because it can be controlled both
genetically and silvicultural and because gains can be
substantial and rapid (Zobel and Talbert 1984). Selection of
plus trees do for changing some characteristics like growth
rate, stem form, resistance to disease, branching habit and
wood structure. It is also provided for reproduction of
desirable characteristics. When this characteristics are
controlled genetically, they can be affected on mean of gain
of selected trees (Mahoney and Fins 2001) but some trees
that have a high growth rate or good stem form do not
always produce industrially desirable wood (Ishiguri et al.
2007). The differences in wood properties among
provenances, families and/or individual trees provide an
opportunity for breeding programmers to select superior
trees for solid wood production (Gapare et al. 2012).

CONCLUSION
This study demonstrates that altitude of about 300 m
could not seriously have effect on fiber morphology and
biometry coefficients in beech trees. Selection of plus trees
without examination of wood properties may be useless for
improving programs. The results from this study suggest
that identification of beech plus trees have to do with
considering phenotype and desirable wood properties
depend on final use. It is necessary to do progeny test to
prove heritage of wood properties to gene conservation
stands as well. also, after they use as reproductive material
for proper use.

INTRODUCTION
Coral disease is defined as an abnormal condition of an
organism that impairs organism functions, associated with
specific symptoms and signs (ICRI/UNEP-WCMC 2010).
It may be caused by external factors, such as infectious
disease, or it may be caused by internal dysfunctions. Coral

disease outbreaks are having a significant, negative impact
on the structure and appearance of coral reefs, and have
contributed to unprecedented declines in live coral cover
and productivity of coral reef ecosystems upon which many
millions of people depend (Galloway et al. 2009). The
same authors concluded that several diseases are playing an
increasingly important role in controlling coral population

36

5 (1): 35-43, May 2013

size, diversity and demographic characteristics.. Large
scale disease outbreaks have already fundamentally altered
the structure of reef communities in the Caribbean (Harvell
et al. 2004). Research on the causes of coral disease has
increased in recent years, especially in terms of identifying
the pathogens involved (Harvell et al. 2007). Most biotic
coral diseases are believed to be related to infection by one
or a group of pathogens (Sokolow 2009). A host of
contributing microorganisms (Richardson and Aronson
2000) and macroparasites such as ciliates (Cróquer et al.
2006) have been identified as possible causal agents;
however, little is currently known about the involvement of
viruses (Sokolow 2009). Research on the causes of coral
disease has increased in recent years, especially in terms of
identifying the pathogens involved (Harvell et al. 2007).
Knowledge of organisms that transmit pathogens from a
reservoir to a host (vectors), the mechanisms by which
coral disease is transmitted between organisms (vector
pathways), and natural reservoirs of coral disease is far
from complete (ICRI/UNEP-WCMC 2010). Growing
evidence suggests that environmental and anthropogenic
stressors are linked with coral disease and mortality in
complex ways (Harvell et al. 2007). Examples of of those
stressors are nutrient enrichment (Garren et al. 2008),
ocean acidification (Sokolow 2009), algal competition
(Aronson and Precht 2006), irradiance (Boyett et al. 2007)
and loss of biodiversity (Keesing et al. 2010).
Coral disease identification is often based on visual
cues observed in the field or from photographs. Such
techniques have been shown to be insufficient for making
coral disease because different causes of disease can result
in similar obvious manifestations of disease, or progress
from showing the signs of one disease to showing those of
another (Ainsworth et al. 2007). Ammar (2012) provided a
guide to coral diseases in the northern Red Sea, Egypt.
Laboratory analyses of samples to identify the microbiological factors accompanying the disease manifestations,
such as the presence or absence of certain pathogens, are
therefore necessary to support accurate disease diagnosis
and accurate disease identifications (Ainsworth et al. 2007).
The purpose of the study is to quantify the coral diseases
in many areas of the Gulf of Aqaba and Ras Mohammed
(South Sinai), Egypt. In addition, the environmental drivers
of disease, as well as understanding the coral’s ability to
resist the disease are studied. A data based on coral
diseases in the area will be established, this will help using
coral diseases as indicators of environmental impacts and
acting to remove or minimize these impacts. Removing or
minimizing these impacts will improve the coral reef
environment, in turn help to increasing fish stocks, tourist
attraction, improving the national income, the economic,
scientific and medical values and conserving the marine
biodiversity.
MATERIALS AND METHODS
Six sites along the Gulf of Aqaba and Ras Mohammed,
Red Sea, Egypt (Figure 1, Table 1) were studied for coral
diseases.

Coral diseases were quantified as percentage cover
relative to the bottom cover. SCUBA diving and the
camera frame (as a quadrat) were used for surveying the
coral diseases. Ten frames, one meter intervals and one
meter from the object were surveyed along a transect fixed
horizontally along the reef contour at the depths reef flat, 1
m, 5 m, 10 m, 15 m, 20 m or till the end limit of coral
growth at each of the studied sites. A FinePix F50, 12
Mega Pixels Digital Camera, was used for taking a series
of underwater photos to help identification of species and
coral diseases. The computer software Photogrid 1.0 beta
Acad was used for ecological analysis of digital
photographs for coral diseases.
Coral disease pathogen identification was achieved
using ICRI/UNEP-WCMC (2010), Raymundo et al.
(2008), Rosenberg et al. (2007).
Disease definition and disease types
Only clear and unequivocal signs of disease were
recorded. Coral disease was also carefully distinguished
from coral bleaching (Brown 1997), which superficially
can look like disease. To make a disease determination,
observers looked for active tissue necrosis. Often this was
accompanied by bared skeleton, mucus production and
partial disintegration of polyps. Blemishes, slight
discolorations and small, cryptic examples of disease were
not scored. We chose characteristics that were as
pathognomicas possible for underwater determinations.
Anchor scrapes, parrot fish bites, predatory snail wounds,
etc were not scored as diseases but as causative agents.
Quality assurance/quality control
After the first survey of sites, the underwater survey
lines were taken up, and then reset and surveyed once
again. In addition, a video tape of lines were done.
RESULTS AND DISCUSSION
Number and percentage cover of coral diseases, healthy
corals and associated biota in each of the studied sites are
shown in Table 2, while the infected coral species by
different diseases are found in Tables 3. Number of coral
diseases ranges from 6 diseases at site 4 (Eeel Garden) to
12 diseases at site 3 (Canyon). However the site having
the lowest number of coral diseases (site 4-6 diseases)

is characterized by the highest percentage cover of coral
diseases (24%) Those diseases are sediment damage, dark
spots, coral neoplasia, ulcerative white spots, coral
hyperplasia and atramentous necrosis. Sites 5 (Shark
Observatory) and 6 (Yolanda) are characterized by the
highest amount of percentage healthy corals (85% and 83%
respectively) while the lowest value is found in site 4
(16%). The highest percentage cover of algae/sea grasses
(22%) is found in site 4 while the lowest percentage cover
of each of algae/seagrasses, macroborers, and sediments is
found in site 5.
The coral disease atramentous necrosis attained the
highest percentage cover in all sites (5, 5, 6, 6, 2 and 3%)
in sites 1-6 respectively. Diseases having lowest percentage
cover are black band disease (site 1), white band disease
(site 2), pigmentation response (site 3), coral hyperplasia
(sites 4, 5) and black band (site 6). A total of 16 diseases
were reported being distributed in the following order in
sites 1-6: 9, 9, 12, 6, 8 and 7 respectively. The coral disease
atramentous necrosis is the most widely distributed one
being found in all 6 sites followed by dark spots and
ulcerative white spots being reported in 5 sites. The disease

that is least distributed is the white tips being reported in
site 5 only. However, each of the black band, white spots,
white band, pigmentation response, sediment damage and
rapid wasting is reported in 2 sites only. The most
commonly distributed disease (atramentous necrosis)
infected six corals in site 1, two corals in site 2, nine corals
in site 3, two corals in site 4, five corals in site 5 and five
corals in site 6. However, the least commonly distributed
disease (white tips) infected only two corals (Acropora
humilis and Millepora dichotoma).
It is observed that the coral disease ulcerative white
spots is always associated with vermetidae predation in
both earlier and later stages of the disease, and in many
cases with Tridacna boring in later stages of the disease.
However, vermetidae predation is also associated with
tissue discolouration (non white pigmentation response)
while Drupella predation is associated with skeletal
eroding band. The coral disease tissue coral neoplasia is
found only in site 4 having 4 percentage cover and
infecting the two coral species Leptoseris incrustans and
Favia speciosa.

Trace metals in water and sediments
Total metal concentrations in seawater varied between
0.1 ppb for Cu and 2.51 ppb for Zn. Site 1, in the north,
recorded the maximum metal concentrations for Zn, Cd, Pb
and Ni . This may be due to the high pollution load from
large cities and harbors like Aqaba and Elat. Site 2, south
to Nuweiba, recorded highest Cu concentration, this may
be due to pollution coming from Nuweiba harbour city.
While, metal concentrations in sediments varied between
1.5 ppm for Cd and 20.79 ppm for Ni. Site 1 recorded the
maximum metal concentrations for Cu, Zn and Pb. Site 4,
in Dahab recorded the highest levels of Cd and Ni (Table 4).
Table 4. Trace elements in surface water and sediments (ppb)
Element Site 1
Site 2 Site 3 Site 4
Water
Cu
0.26
0.28 + 0.22
0.13
Zn
2.51+
1.98
1.03
0.96
Cd
1.56 +
1.11
0.73
0.72
Pb
1.11 +
0.87
0.71
0,77
Ni
0.87 +
0.66
0.57
0.49
Sediment
Cu
4.67 +
3.97
3.72
3.63
Zn
11.33 + 7.27
6.25
6.71
Cd
3.57
3.27
3.43
3.89 +
Pb
17.74 + 17.55 16.76 16.91
Ni
17.23
16.49 19.25 20.79 +
Note: *Rocky bottom with no sediments

Site 5

Site 6

0.11
0.72
0.66
0.61
0.39

0.09
0.68
0.61
0.57
0.41

2.92
5.28
1.49
12.46
13.58

*
*
*
*
*

Discussion
As it is obvious from the present study, the site having
the lowest number of coral diseases (site 4) is characterized
by the highest percentage cover of coral diseases indicating
space monopolization and outbreak of those diseases. The
same site is characterized by the highest percent cover of
sediments, suggesting that sedimentation may increase the
percent coral diseases at the expense of disease number.
This could be due to decreased coral mortality with
increased sedimentation, decreasing the available substrate
or space for diverse diseases. Decreased resistance of the
host coral caused by adverse environmental conditions may
increase opportunistic diseases (Harvell et al. 1999) leading
to increased coral mortality (Haapkyla et al. 2009). Changes
in the population size (e.g. percentage cover), growth and
reproduction of a communityâ&#x20AC;&#x2122;s primary producers (e.g.
algae) and major framework builders will have impacts on
the community. These changes are especially relevant
given the longevous age structure of corals and, as
compared to macroalgae, their relatively slow coral
recruitment (Tougas and Porter 2002). This agrees with the
results of the present study in which the highest percentage
cover of algae/sea grasses is associated with the highest
percentage cover of coral diseases (site 4), but the lowest
percentage cover of each of algae/seagrasses, macroborers,
and sediments is associated with the lowest percentage
cover of coral diseases (site 5).

Jones et al. (2004) indicated that, Fluorescence in situ
hybridisation (FISH) techniques and cloning, and analysis
of the 16S rRNA genes from diseased coral tissue infected
with atramentous necrosis, identified a mixed microbial
assemblage in the diseased tissues particularly within the
Alphaproteobacteria, Firmicutes and Bacteroidetes. In the
present study, the presence of the coral disease atramentous
necrosis attaining the highest percentage cover and
infecting the highest number of corals in all sites, is
associated with vermetidae predation in site 1, Tridacna
boring, vermetidae in site 2, mechanical breaking,
vermetidae in site 3, filamentous algal overgrowth in site 4,
mechanical breaking, vermetidae in sites 5 and 6. This is an
evidence that vermitidae predation, Tridacna boring and
mechanical breaking may evoke growth of Alphaproteobacteria, Firmicutes and Bacteroidetes, promoting the
growth of filamentous algae. Outbreak of the coral disease
atramentous necrosis may be greately attributed to the
terrestrial runoff caused by higher rainfall and in turn,
decreased salinity (Harvell et al. 1999). This may have lead
to increased stress on corals that may have reduced their
immune responses, and/or increased virulence of pathogen
(s) causing the disease. Decreased resistance of the host
coral caused by adverse environmental conditions may also
increase opportunistic diseases (Harvell et al. 1999).
Results of the present study showed that the coral disease
atramentous necrosis attained the highest percentage cover
in all sites. This result, together with the fact that the
present sites lie all in wadis being liable to terrestrial runoff
, make the present results in aggrement with that of Harvell
et al. (1999).
Lesions with signs that are similar to ulcerative white
spots (UWS) can be caused by fish bites. Parrotfish lesions
can be distinguished by the presence of skeletal damage,
while the tubelip wrasse, Labrichthys unilineatus will
remove tissue without damaging the skeleton. Arboleda
and Reichardt (2010) stated that, the causative agent of the
Indo-Pacific coral disease, Porites ulcerative white spot
syndrome (PUWS), that affects Porites spp. and a few
other coral genera has so far remained unidentified. In the
present study, the association of the coral disease ulcerative
white spots with vermetidae predation in both earlier and
later stages of the disease, and in many cases with Tridacna
boring in later stages of the disease is an evidence that
vermetidae is a causative agents of the disease but
Tridacna spp. could take the disease as a suitable substrate.
In the present study, this disease infected Porites solida,
Goniastraea retiformis, Favites flexusa and Favia speciosa.
Wooldridge (2010) indicated that coral's failure to
prevent the division of zooxanthellae leads to ever-greater
amounts of the photosynthesis-derived carbon to be
diverted into the algae rather than the coral. This makes the
energy balance required for the coral to continue sustaining
its algae more fragile, and hence the coral loses the ability
to maintain its parasitic control on its zooxanthellae leading
to bleaching. In the present study, the white tips, which is
somekind of bleaching, and infecting only the two species
Acropora humilis and Millepora dichotoma, indicates that
those two species are the most sensitive species that loses
the ability to maintain its parasitic control on its

41

zooxanthellae. Lesions of tissue discolouration (non white
pigmentation response) may be caused by borers,
competitors, algal abrasion, fish bites, breakages, etc
(Beeden et al. 2008). In the present study, the non white
pigmentation response was associated with vermitidae
predation) while skeletal eroding band was associated with
Drupella predation.
Yamashiro et al. (2000) found coral neoplasia to be
associated with the global coral bleaching event (1998). In
the present study, the coral disease tissue coral neoplasia,
being found only in site 4, was associated with high
sediment load, low salinity due to fresh water coming from
the adjacent tourist showers which are just close to the shore.
Sites 5 and 6, having the most healthy, rich, and nice
reef slopes in the Red Sea, have their diseases restricted
only on the reef flat. Those diseases of the reef flat are
associated with mechanical breaking (due to trampling on
the reef flat), vermetidae predation, gastropod boring,
aggressive coralline algal overgrowth, Drupella predation
and Parrot fish predation.
Diseases having lowest percentage cover are black band
disease (site 1), white band disease (site 2), pigmentation
response (site 3), coral hyperplasia (sites 4, 5), black band
disease (site 6). However, Richardson (1998) indicated that
the incidence and prevalence of black band disease may
also increase when corals are stressed by sedimentation,
nutrients, toxic chemicals and warmer-than-normal
temperatures. Histopathological examinations of diseased
tissue of white band disease (WBD) revealed basophilic
ovoid bodies up to 40 Îźm (Peters et al. 1983). Electron
microscopy of thin sections of the ovoid bodies revealed
that they were composed of Gram-negative bacteria ,
suggesting that these bacteria may be the causative agent of
the disease. Pigmentation response is considered a response
of the coral host to a variety of stressors (e.g. unidentified
pathogens, competition, predation, boring fauna, abrasion,
etc.), suggesting that organism health is compromised
(Raymundo et al. 2008).
White plague was reported in sites 3, 6. Richardson
(1998) succeeded in isolating from diseased corals with
white plague, a new species of Sphingomonas that infected
healthy corals in laboratory experiments. Although the
mechanism trigerring coral neoplasia or tissue coral
neoplasia is still unknown and thought to be a genetic
mutation that may be the result of environmental conditions
(Yamashiro et al. 2000), tissue coral neoplasia disease in
the present study ,being reported only in site 4, was
associated with high sediment load, low salinity due to
fresh water coming from the adjacent tourist showers which
are just close to the shore. The disease in the present study
was recognized as slightly hemispherical protuberances
with fewer numbers of polyps per surface area, fewer
zooxanthellae per polyp, finer skeletal structures than
normal and reduced fecundity in coral neoplasia areas.
Infected corals relative to water and sediment quality
As human populations continue to increase, nutrients,
terrigenous silt, pollutants and even pathogens themselves
can be released into nearshore benthic communities
(Raymundo et al. 2008). It was further discussed in that

42

Â

5 (1): 35-43, May 2013

book that while the link between anthropogenic stress and
disease susceptibility is currently poorly understood, one
hypothesis is that coral disease is facilitated by a decrease
in water quality, particularly due to eutrophication and
sedimentation. Growing evidence suggests that environmental and anthropogenic stressors are linked with coral
disease and mortality in complex ways (Harvell et al.
2007). Like, many benthic filter feeders, corals assimilate
differentially certain amounts of solid metals, mainly
through ingestion of contaminated particles (Madkour
2011). Ammar et al. (2005) concluded that the toxic effect
of a certain metal on a coral may have the growth rate, in
turn skeletal density, decreased with increasing metal
concentration. This may foster the infection of the coral
with a certain disease as well. Site 1 has 9 diseases
infecting 12 coral species, of which Cyphastrea serialia is
infected with highest number of diseases (atramentous
necrosis, dark spots disease, skeletal eroding band and
white plague). Site1 is characterized by the maximum
metal concentrations of Zn, Cd, Pb and Ni in water and
highest metal concentrations for Cu, Zn and Pb in
sediments due to the high pollution load from large cities
and harbors like Aqaba and Eilat. Site 2 has 9 diseases
infecting 10 corals, of which Millepora dichotoma is
infected with the highest number of diseases (white plague,
atramentous necrosis, brown band disease and coral coral
hyperplasia). Site 2 is characterized by the highest Cu
concentration in water due to pollution coming from
Nuweiba harbour. Site 3 has 12 diseases infecting 19
species (highest number of all sites), of which Porites lutea
is infected with the highest number of diseases
(atramentous necrosis, brown band disease, white band
disease and skeletal eroding band). Site 4 has 6 diseases
infecting 11 species, each of which is infected with only
one disease except Favia speciosa and Goniastrea
retiformis which are infected with 2 diseases for each one.
Those fewer number of coral diseases in site 4 attained the
highest percentage disease cover indicating space
monopolization of those six diseases. This site attaind the
the highest levels of Cd and Ni in sediments and highest
percentage sediments. Site 5 has 8 diseases infecting 10
species, of which Stylophora pistillata is infected with the
highest number of diseases (atramentous necrosis, pigmentation response, dark spots disease and coral hyperplasia).
Site 6 has 7 diseases infecting 8 species, of which Porites
solida is infected with the highest number of diseases
(ulcerative white spots, black band disease, white plague
and white patches). Sites 5 and 6, having low number and
lowest percentage cover of coral diseases, are characterized
by lowest levels of trace elements in both water and
sediments. They are also characterized by nice and ideal
slopes. Their coral diseases were reported only on the reef
flat, probably because the reef flat is sheltered, lying below
high mountains, a condition that may promote bacterial
growth on the reef flat.

percentage cover of algae/sea grasses is associated with the
highest percentage cover of coral diseases. The coral
disease atramentous necrosis attained the highest
percentage cover in all sites which lie all in wadis being
liable to terrestrial runoff. There is an evidence that
vermetidae predation could be a causative agents of
ulcerative white spots but Tridacna spp. could take the
disease as a suitable substrate. The coral disease tissue
coral neoplasia, being found only in site 4, was associated
with high sediment load, low salinity due to fresh water
coming from the adjacent tourist showers which are just
close to the shore. Site 1 has 9 diseases infecting 12 coral
species, of which Cyphastrea serialia is infected with
highest number of diseases (atramentous necrosis, dark
spots disease, skeletal eroding band and white plague). This
site is characterized by the maximum metal concentrations
of Zn, Cd, Pb and Ni in water and highest metal
concentrations for Cu, Zn and Pb in sediments. Site 2 has 9
diseases infecting 10 corals, of which Millepora dichotoma
is infected with the highest number of diseases (white
plague, atramentous necrosis, brown band disease and coral
hyperplasia). This site is characterized by the highest Cu
concentration in water. The fewer number of coral diseases
in site 4 attained the highest percentage disease cover
indicating space monopolization of those diseases. This site
attaind the the highest levels of Cd and Ni in sediments and
highest percentage sediments. Sites 5 and 6, having low
number and lowest percentage cover of coral diseases, are
characterized by the lowest levels of trace elements in both
water and sediments.
Identifying knowledge gaps that impede understanding
coral disease mechanisms, and limiting elucidation of
causes, significance or control of coral disease.
Recommending directed research and education to fill
these knowledge gaps. Standardizing methods for
investigating coral disease outbreaks considering both
biotic and abiotic etiologies. Addressing issues relative to
the management of coral reef resources; and fosters
collaboration among partners, stakeholders, key marine
resource management agencies, and regional networks.
Developing guidance for the proper handling and
containment of corals in infectious disease experiments.
Fostering the development of a cohesive coral disease
research community.

Sedimentation may increase the percent coral diseases
at the expense of disease number. However, the highest

ACKNOWLEDGEMENTS
This work was done in the frame of the strategy of the
National Institute of Oceanography and Fisheries "Coral
disease distribution as biomonitors of environmental
impacts" for the year 2011/2012.
REFERENCES

Abstract. Pyasi A, Soni KK, Verma RK. 2013. Effect of ectomycorrhizae on growth and establishment of sal (Shorea robusta) seedlings
in central India. Nusantara Bioscience 5: 44-49. The aim of the present study was to develop ectomycorrhiza in sal sapling at outside
the sal growing areas. For this purpose sal seedling were raised at Jabalpur which is around 80 km away from natural sal forest
(Motinala, MP). Seed sowing was done with inoculation of ectomycorrhizal inocula prepared by isolating the fungi from surface
sterilised young basidiocarp of Lycoperdon compactum and Russula michiganensis. The inocula of ectomycorrhizal fungus were
prepared in wheat grains treated with gypsum. The synthesis of ectomycorrhiza was observed in the sapling planted in the experimental
field at Jabalpur with production of basidiocarp of Lycoperdon compactum near saplings. The mycorrhized saplings also showed higher
growth indices.
Key words: ectomycorrhizal inoculum, ectomycorrhiza synthesis, nutrient uptake, sal forest

INTRODUCTION
Sal (Shorea robusta C.F. Gaertn.; Figure 1)
is one of the most important sources of
hardwood timber in India. Density of sal forest
has significantly reduced from sal dense 65.6%
in 1976 to 11.1% in the year 1999 followed by
sal open 11.2% and sal medium 18.2%. The
overall change has been estimated to be 42.1%
of the total forested area (Chauhan et al. 2003).
Decreasing sal forest cover is one of the top
ranked problems of forest department. The
primary reason for decreasing it so rapidly is
poor regeneration. The seed of sal is
recalcitrant, and start to germinate just before it
detaches from the tree. It immediately needs
appropriate moisture, nutrient and mycorrhiza
for its establishment. A mycorrhiza in general is
a symbiotic relation between a fungus and the
roots of a vascular plant. Ectomycorrhization
refers to the infestation of cortical tissues of
root by hyphae of mycorrhizal fungi (Harley

11959). Mycorrrhizae form a mutualisticc relationship with
t roots of most
the
m plant speccies. This mutuualistic associiation
p
provides
the fungus with relatively coonstant and direct
d
a
access
to carrbohydrates, such as gluucose and suucrose
s
supplied
by thhe plant. Thee carbohydratees are transloocated
f
from
their souurce to root tissue and on to fungal partneers. In
r
return,
the plaant gains the benefits
b
from the mycobionnts in
t
terms
of wateer and mineraal nutrients thhus improvinng the
p
plant's
mineraal absorption capabilities. Plant roots alone
m be incappable of takinng up phosphhate ions thaat are
may
d
demineralised
, for examplee, in soils witth a basic pH. The
m
mycelium
of the
t mycorrhizzal fungus cann, however, access
a
t
these
phosphoorus sources, and
a make theem available to
t the
p
plants
they coolonize (Boween et al. 19744). The absennce of
m
mycorrhizal
f
fungi
can alsso slow plantt growth in early
s
succession
or on degradedd landscapes. Fungi have been
f
found
to have a protective roole for plants rooted in soilss with
h
high
metal cooncentrations, such as accidic contaminated
s
soils
and coal mine restoratiion (Bauman et al. 2013).
Ectomycorrrhiza play an
a important role in sal forest
f
e
ecosystem
as it
i forms mutuaalistic accociaation with a vaariety
o basidiomyyceteous and gasteromyceeteous fungi. The
of
m
main
ectomyccorrhiza forming fungi in central India have
a
already
been studied and reported
r
of which
w
the com
mmon
f
fungi
are: Astrraeus hygrom
metricus, Boleetus edulis, Booletus
f
fallax,
Geasstrum tripleex, Lycoperddon compactum,
and
S
Scleroderma
bovista,
Sclerodermaa
geaster
verrucosum, Russula adusta, Ruussula
S
Scleroderma
c
cinerella,
Russsula deliculaa, Russula leelavathyi, Ruussula
m
michiganensis
s (Bakshi 1974, Soni et al. 2011, Pyasi et al.
2
2011,
2012). The
T basidiocaarp of ectomyycorrhizal funggi are
p
produced
on soil surface inside sal foorest. The acttively
g
growing
basiddiocarp helps young seedllings in absoorbing
n
nutrients
mainnly the phosphhorous and othher trace elem
ments.
T life of bassidiocarp is very short usuaally 2-3 days but it
The
p
production
caan persist for more than 3 consecutive rainy
m
months.

4
45

In
n present stuudy isolatatioon of two ectomycorrhizaal
fung
gi in pure culture
c
were made and used them to
t
synth
hesise ectomyycorrhiza in saal sapling plan
nted in non-saal
foresst area.
TERIALS AN
ND METHOD
DS
MAT
Loca
ation of experriment
The
T experimennt was conduccted at researcch experimentaal
area of Forest Pathology Division, Trropical Forest
Reseearch Institutte, Jabalpur,, Madhya Pradesh,
P
Indiia
(Figu
ure 2) which is
i located on N 23006’074”” E 79059’386”,
elevaation above seea level 415 m
m. It is aroun
nd 80 km awaay
from
m natural sall forest. Thee place expeeriences warm
m
weatther during Appril-June withh average temp
perature rangees
betw
ween 41oC-21oC during thhese months and 27oC-8oC
durin
ng the wintters (Novembber-February). The annuaal
rainffall is 1386 mm,
m and monssoon arrives at
a this place in
i
the beginning
b
of July
J
and prolonng up to Septeember.
Isola
ation of ectom
mycorrhizal ffungi and preeparation of
inocula
Fresh
F
and tendder fruit bodiees of Lycoperd
don compactum
m
G.H.. Cunn. andd Russula m
michiganensis Shaffer waas
colleected from saal forest and washed undeer running taap
wateer for five minnutes. After thhat it was placced in aqueouus
soluttion of sodiuum hypochloorite, 0.1% (w/v
(
availablle
chlorrine) for ten minutes and then the basidiocarps werre
cut into
i
small annd thin slices using a sterile razor blade.
Thesse sections were
w
again flloated into double
d
distilleed
autocclaved water to remove ddisinfectants. These sectionns
weree inoculated onto
o
Norkranss and PDA meedia containinng
a traace of Bavistiin (R) and Streeptomycin su
ulphate in Pettri
dishees (Figure 3D
D) and incubatted at 25-270C.
C After growtth
the fungi
f
were trannsferred to cuulture tubes (Figure 3E).

Table 1. Growth indices of sal saplings treated with different
organic amendments and cultures of ectomycorrhizal fungi after 9
months of planting in micro-plots.

Sal Litter

Observation and recording of data
The basidiocarp of ectomycorrhizal fungi are produced
on soil surface near inoculated seedlings. The life of
basidiocarp was recorded 2-3 days but it production persist
for more than 3 consecutive months of rainy season.
Basidiocarp of Lycoperdon compactum are produced in
close vicinity of roots in artificially inoculated sal saplings
during July-August.
Heights of saplings, leaf breath and length were
measured using standard centimetre scale. Diameter of
saplings at collar region was measured using Varner's
callipers. Leaf area index was calculated by multiplying
maximum length of leaf with breadth (cm2). Growth
indices of seedlings were calculated after 9 months of
planting using the following formula:

Results
Basidiocarps of Lycoperdon compactum were observed
in groups of 1-3 near ECM2 treated saplings in two blocks
in close vicinity forming ectomycorrhizal association with
feeder roots of sal at Jabalpur (Figure 3A-B). Mycorrhiza
developed by the winter season (Figure 4A-B). No
basidiocarp was observed in control and other treatments.
Basidiocarps produced are short lived usually 2-3 days
which upon maturation releases basidiospores. At Jabalpur
basidiocarp was produced 3 times during July-August 2012
when rainy season was at its peak. The basidiocarp is
rough, tough, globose, yellowish pinkish brown 2-5 cm in
diameter, a typical gasteromyceteous shape, with a small
rhizoidal stalk at base. Young sporophore is hard,
depressed above, exoperidium glabrous, smooth 1-1.5 mm
thick leathery, endoperidium smooth very thin. Gleba
olivaceous, amber when young, becomes powdery
chocolate brown at maturity. Spores abundant, round,
hyaline to olivaceous brown, verrucose 4.5-5.0 µm (Figure
3C). The fungus grows on PDA agar medium with milky
white, irregular colonies, rhizoidal, radiate, slow growing
35 mm. in diam after 7 days at 25±20C.
The maximum growth indices was observed in
vermicompost+ECM2 treatment, which was 3.4 times more
than control followed by ECM2, 3.0 times more,
vermicompost+ECM1, 2.1 times more, sal litter+ECM2,
2.1 times more and sal litter+ECM1, 1.8 times more. Other
treatments have no significant effect on growth indices of
sal saplings (Table 1).

ECM1

Inoculation and planting of seedlings
The experimental seedlings received the following
treatment and are arranged in CRD on a cemented
platform, i.e. (i) Control, (ii) Sal litter, (iii) ECM1 (Russula
michiganensis), (iv) Sal litter+ECM1, (v) Vermicompost+
RCM1, (vi) ECM2 (Lycoperdon compactum), (vii) Sal
litter+ECM2, (viii) Vermicompost+ECM2, and (ix)
Vermocompost.
Sal litter collected from sal forest of Motinala, Balaghat
District, Madhya Pradesh, India (Figure 2) and added to the
local soil in the ratio of 1:4 (v/v). The vermicompost was
also mixed in the same ratio. The potting mix was filled in
black polyethylene bags of 7x9". Ten g inocula of
ectomycorrhizal fungi were given to each seedling of
treatment numbers 3-8. The inoculum was placed just
below seeds during seed sowing in pre monsoonal period.
After five months the seedlings were planted in the microplots with 1x0.75m spacing at the Institute campus in
RCBD. Four blocks each with all the above mentioned
treatments were made. The seedlings were watered with
tube well water as and when required.

RESULTS AND DISCUSSION

Control

For preparation of Ectomycorrhizal inocula the culture
isolated as above were multiplied in water soaked boiled
wheat grains treated with gypsum salt and a pinch of
Bavistin in a narrow mouth glass bottles (Figure 3F). Six to
eight mycelial agar discs of mycorrhizal fungi were
inoculated in to the wheat grains. After 10-12 weeks of
incubation at 25-270C the inoculum was ready for use
(Kanan and Natrajan 1988).

Discussion
Mycorrhiza is a beneficial association between fungi
and root of higher plants. About 85 percent higher plants
(gymnosperms and angiosperms) form this type of
relationship in forests (Bakshi 1974). In fact this
sustainable relationship is a deal between mycobionts and
phycobionts for their survival and to some extent it may
prove to be an obligatory for both the partners. This

beneficial relationship can be exploited in establishing new
plantations at deteriorating sites and in degenerating forest
covers. In the present study we established an
ectomycorrhizal synthesis in vivo by inoculating two
ectomycorrhizal fungi during seed sowing along with other
treatment like sal litter and vermicompost which provides
sal seeds the natural consortium of microbes which support
its growth. But these natural consortiums of microbes when
used alone are not very much effective in growth of sal
saplings. These are more effective when used along with
the cultures of ectomycorrhizal fungi. Among the two
ectomycorrhizal fungi used only Lycoperdon compactum
produces basidiocarps in short duration. Another species
Lycoperdon perlatum and Russula parasurea are reported
to form mycorrhiza with P. patula in Nilgiri Biosphere
Reserve Tamil Nadu, India (Mohan 1991). In Malaysia two
another dipterocarps also developed ectomycorrhiza with
locally isolated ectomycorrhizal fungi (Lee et al. 2008).
Many scientists from India and abroad have artificially
established mycorrhization in Pinus and Eucalyptus spp.
and have got fruitful results (Alexander 1981; Marx and
Ross 1970). In central India, Sharma et al. (2009) have
synthesise ectomycorrhiza in a monocot host,
Dedrocalamus strictus with Cantharellus tropicalis. In our
study we used wheat grains for production of inocula of
ectomycorrhizal fungi similarly Kannan and Natrajan
(1988) also produced spawn of Laccaria laccata and
Amanita muscaria but in sorghum grains. In the present
study we have tried sal litter collected from natural sal
forest to inoculate the seedling in non-sal area similarly
Lakhanpal (1987) used natural roots of mycorrhizal trees
for artificial inoculation of Pinus gregardiana and Picea
smithiana seedlings to develop mycorrhiza.

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